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Method Article
Paired-end analysis of transcriptional start sites
Ting Ni
Duke University
Corresponding Author
License:
This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License
We describe an improved deep sequencing strategy, paired-end analysis of transcriptional start sites (PEAT), to globally characterize transcriptional start sites (TSSs) in a previous study [1]. It was used to explore the landscape of transcription initiation in Drosophila melanogaster embryo. Here we present a detailed protocol, with minor modifications, can be broadly applied to investigate capped transcripts in other eukaryotes, from budding yeast to rat and human cell lines.
Keywords
Transcriptional start sites, Paired-end sequencing, Initiation motif, Internally capped transcripts
Figure 1
Figure 2
Figure 3
TRIzol reagent (Invitrogen, cat. no. 15596-018)
Nuclease-free Water (Ambion, cat. no. AM9932 )
RNaseZap RNase Decontamination Solution (Ambion, cat. no. AM9780)
Phenol:Chloroform:Isoamyl Alcohol (25:24:1), pH8.0 (Sigma, cat. no. P2069)
Chloroform (VWR, cat. no. MK444010)
Glacial acetic acid (VWR, cat. no. 87003-238)
2-Propanol (Sigma, cat. no. I9516)
Absolute ethanol (VWR, cat. no. 200004-482)
Formaldehyde solution (Sigma, cat. no. F8775)
Trizma hydrochloride (Sigma, cat. no. T5941)
Trizma base (Sigma, cat. no. T6066)
Sodium chloride (Sigma, cat. no. S3014)
Sodium acetate (Sigma, cat. no. S7670)
EDTA (Sigma, cat. no. E5134)
SDS (Sigma, cat. no. L4390)
Boric acid (Sigma, cat. no. B7901)
Urea (Sigma, cat. no. U0631)
Lithium chloride (Sigma, cat. no. L9650)
Lithium dodecyl sulfate (Sigma, cat. no. L9781)
Triton X-100 (Sigma, cat. no. T8787)
DMSO (Sigma, cat. no. D8418)
Agarose (Bio-Rad, cat. no. 161-3102EDU)
Ethidium Bromide Solution (Bio-Rad, cat. no. 161-0433)
40% acrylamide and bis-acrylamide solution, 19:1 (Bio-Rad, cat. no. 161-0144)
Ammonium Persulfate (Bio-Rad, cat. no. 161-0700)
TEMED (Bio-Rad, cat. no. 161-0800)
RNeasy Mini kit (Qiagen, cat. no. 74104)
RNeasy MinElute Cleanup Kit (Qiagen, cat. no. 74204)
RNase-Free DNase Set (Qiagen, cat. no. 79254)
Dynabeads Oligo(dT)25 (Invitrogen, cat. no. 610-02)
SuperScript III Reverse Transcriptase (Invitrogen, cat. no. 18080-093)
Bacterial Alkaline Phosphatase, E.coli C75 (Takara, cat. no. TAK 2120B)
Ampligase Thermostable DNA Ligase (Epicentre, cat. no. A32750)
Tobacco Acid Pyrophosphatase (Epicentre, cat. no. T19100)
T4 RNA Ligase 1 (NEB, cat. no. M0204L)
Phusion High-Fidelity DNA Polymerase (NEB, cat. no. F-530L)
Taq DNA Polymerase (NEB, cat. no. M0273L)
Exo I nuclease (NEB, cat. no. M0265S)
Exo III nuclease (NEB, cat. no. M0206S)
Phi29 DNA polymerase (NEB, cat. no. M0269L)
MmeI (NEB, cat. no. R0637L)
XhoI (NEB, cat. no. R0146M)
RNasin ribonuclease inhibitor (Promega, cat. no. N2515)
dNTP Mix (Bioline, cat. no. BIO-39029)
HyperLadde I DNA ladder (Bioline, cat. no. BIO-33026)
HyperLadde IV DNA ladder (Bioline, cat. no. BIO-33030)
10 bp DNA Ladder (Invitrogen, cat. no. 10821-015)
Ribonuclease H (Invitrogen, cat. no. 18021-014)
OptiKinase (USB, cat. no. 78334Y)
Actinomycin D (USB, cat. no. 10415)
GlycoBlue (Ambion, cat. no. AM9516)
ZYMO DNA clean & concentrator-5 kit (ZYMO, cat. no. D4014)
PEG 8000 (VWR, cat. no. PAV3011)
Biotin (Long arm) Hydrazide (VWR, cat. no. 101098-478)
ATP solution (VWR, cat. no. 100216-332)
pGEM-T Easy Vector System II (Promega, cat. no. A1380)
1.7 ml microcentrifuge tube (Denville Scientific, cat. no. C2170)
PCR tube strips and cap strips (Axygen Scientific, cat. no. PCR-0208CPC)
Barrier tips, 10 ul (Denville Scientific, cat. no. P1096-FR)
Barrier tips, 20 ul (Denville Scientific, cat. no. P1121)
Barrier tips, 200 ul (Denville Scientific, cat. no. P1122)
Barrier tips, 1250 ul (Denville Scientific, cat. no. P1126)
Microcentrifuge (Eppendorf Model 5424)
Microcentrifuge (Eppendorf Model 5415R)
Pellet pestles blue polypropylene (Sigma, cat. no. Z359947)
Razor Blades (VWR, cat. no. 55411-050)
NanoDrop 1000 Spectrophotometer (Thermo Scientific, cat. no. SID-10135606)
Standard PCR machine (e.g., PTC-200 Thermo Cycler, MJ Research)
Qubit Fluorometer (Invitrogen, cat. no. Q32857)
PCR-Cooler (Eppendorf, cat. no. 022510509)
UV Stratalinker 2400 (Stratagene)
AC600 PCR workstation (AirClean system)
Dynal MPC-T4 Magnet (Invitrogen)
Tube Rotator (VWR, cat. no. 13916-822)
Mini-vertical gel electrophoresis unit (Hoefer, cat. no. SE250)
Sturdier vertical electrophoresis unit (Hoefer, cat. no. SE410)
Standard heating block (e.g., Analog Dry Block Heater, VWR)
Standard water bath (e.g., Analog and Digital Unstirred Water Baths, VWR)
Gel imaging systems (e.g., AlphaImager, Alpha Innotech)
Standard power supply (e.g., PowerPac HC Power Supply, Bio-Rad)
Preparation of RNA samples
Timing: 1h
The quality of the starting RNA samples is critical for the success of the PEAT strategy. We recommend TRIzol reagent to isolate total RNA from higher eukaryotes (both animal and plant). For yeast cells, hot-phenol approach1 tends to give the most reproducible results. To avoid potential biases that may incur during library construction, it is preferable to start with ~150 μg total RNA if the sample is easy to be obtained. For low-abundance samples, it is possible to reduce the amount to 10 μg. In this protocol, we use Drosophila embryos to demonstrate the PEAT strategy. One can adapt the protocol to construct PEAT libraries for other species as well
RNA cleanup
Timing: 1 h
RNeasy Mini kit (QIAGEN) is used to remove genomic DNA contamination in RNA preparation. On-column DNase I digestion step is included according to the manufacturer’s protocol Starting with ~150 μg total RNA, the final product is eluted with 80 μl nuclease-free water.
(Optional Step) The integrity of the purified RNA is checked with1.2% formaldehyde agarose gel (FA gel). Alternatively, Agilent RNA Nano Labchip can be used to determine the RNA quality. Proceed to the next step if the RNA samples pass quality control.
PolyA+ RNA selection with Dynabeads (dT)25
Timing: 3 h
Perform two rounds of polyA+ selection with Dynabeads (dT)25 according to manufacturer’s protocol. Use all of the purified total RNAs from the previous step and elute the polyA+ RNA with 30 μl elution buffer (10mM Tris-HCl, pH7.5).
Lithium salt resulting from polyA+ selection step might affect downstream enzymatic activity. RNeasy MinRlute kit is therefore used to remove residual lithium by following the manufacturer’s protocol. We recommend to elute the column twice, each with 40 μl nuclease-free water. The resulting products are then quantified with a nanodrop spectrophotometer. From 150 μg total RNA, the typical yield of purified polyA+ RNA is 2-3 μg.
Bacterial Alkaline Phosphatase (BAP) treatment
Timing: 2.5 h
- Preparing the following reaction in an RNase-free 1.7ml tube: RNA from step 5 80 μl
BAP enzyme (Takara) 6 μl
10x BAP buffer (Takara) 10 μl
RNasin ribonuclease inhibitor (Promega) 2.5 μl
Nuclease-free water 1.5 μl
Total reaction volume 100 μl
- Incubate the reaction at 37 °C for 45 min. Add 100 μl nuclease-free water to the reaction. Extract once with 200 μl phenol/chloroform and then with 200 μl chloroform. Perform ethanol precipitation by adding 20 μl 3 M NaOAc (pH 5.2), 2 μl GlycoBlue and 500 μl ethanol. Wash the pellet once with 70% ethanol, followed by adding 85.5 μl nuclease-free water to dissolve RNA. Critical step: Completely removal of the phosphate group on the uncapped RNA molecules before TAP treatment is critical for reducing background noise.
Tobacco Acid Pyrophosphatase (TAP) treatment
Timing: 2.5 h
- Preparing the following reaction in a RNase-free 1.7 ml tube: BAP treated RNA from step 7 85.5 μl
10x TAP buffer (Epicentre) 10 μl
TAP enzyme (Epicentre) 2 μl
RNasin ribonuclease inhibitor (Promega) 2.5 μl
Total reaction volume 100 μl
Incubate the reaction at 37 °C for 1 h. Add 100 μl nuclease-free water to the reaction. Extract once with 200 μl phenol/chloroform and then with 200 μl chloroform. Perform ethanol precipitation by adding 20 μl 3 M NaOAc (pH 5.2), 2 μl GlycoBlue and 500 μl ethanol. Wash the pellet once with 70% ethanol. Dissolve the BAP and TAP treated RNA in 13 μl nuclease-free water.
Ligation with 5’ chimeric linker
Timing: 3 h (or overnight)
- Combing the following in a 1.7ml RNase-free tube: BAT/TAP treated polyA+ RNA from step 9 13 μl
30 μM PAGE purified Mme_CAP_RDhyb (IDT) 2 μl
10x T4 RNA ligase buffer (NEB) 10 μl
40% PEG8000 (0.2 μM filtered and autoclaved) 62.5 μl
RNasin ribonuclease inhibitor (Promega) 2.5 μl
T4 RNA ligase1 (NEB) 10 μl
Total reaction volume 100 μl
Critical step: The ratio of linker:RNA should be larger than 10:1 to ensure efficient ligation. The sequence of Mme_CAP_RDhyb: 5’-CTC AAG CTT CTA ACG ATG TAC GCT CG rArGrUrCrCrArArC-3’, ‘r’ denotes ribonucleotide.
Mix the reaction well by pipetting and incubate at room temperature for at least 3 h (or overnight). Add 100 μl nuclease-free water to the reaction and extract once with 200 μl phenol/chloroform and a second time with 200 μl chloroform.
Removal of excess linkers
Timing: 0.5 h
The supernatant from Step 11 is further purified with RNeasy MinElute kit (QIAGEN) to remove excess linkers by following the manufacturer’s protocol. Elute linker-ligated RNA with 20 μl nuclease-free water.
Reverse transcription with random primer
Timing: 2 h
- Prepare the reaction in a PCR tube: RNA sample from previous step 20 μl
10mM dNTP, each (Bioline) 2 μl
10uM Mme_RTN6 (IDT) 2 μl
Incubate the reaction for 5 min at 65 °C, then chill on ice for ≥ 1 min. Add the following:
5x first strand buffer (Invitrogen) 8 μl
0.1M DTT (Invitrogen) 2 μl
RNasin ribonuclease inhibitor (Promega) 2 μl
20x Actinomycin D (USB) 2 μl
SuperScript III reverse transcriptase (Invitrogen) 2 μl
Total reaction volume 40 μl
The sequence of Mme_RTN6: 5’-GCG GCT GAA GAC GGC CTA TCC GAC NNN NNN-3’
Mix and incubate the reaction at 25 °C for 5 min, 50 °C for 1 h, 70 °C 15 min. Hold at 10 °C.
- Add 1 μl RNase H (2 Units, Invitrogen) to the reaction and incubate at 37 °C for 20 min. This treatment helps to remove RNA strand from RNA:DNA hybrid. Use ZYMO DNA clean & concentrator-5 kit to purify the samples following manufacturer’s protocol. Elute with 20 μl nuclease-free water. TIP: Add at least 7-fold of the binding buffer to efficiently recover single-stranded cDNA.
Quality control step 1
RT-PCR is used to evaluate the efficiency of linker ligation.
Timing: 2 h
- Prepare the following in a PCR tube: Purified cDNA from step 15 0.25 μl
5x HF buffer (NEB) 4 μl
10 μM Mme_F (IDT) 1 μl
10 μM Mme_R or fly_GAPDH1_R (IDT) 1ul
10 mM dNTP, each (Bioline) 0.4 μl
Phusion high fidelity DNA polymerase (NEB) 0.2 μl
Nuclease-free water 13.15 μl
Total reaction volume 20 μl
- Perform the thermal cycling below: 98 °C for 30 sec; 30 cycles of 98 °C for 10 sec, 67 °C for 10 sec and 72 °C for 90 sec; 72 °C for 10 min. Sequences of PCR primers: Mme_F: 5’-CTC AAG CTT CTA ACG ATG TAC GCT CGA-3’; Mme_R: 5’-GCG GCT GAA GAC GGC CTA TCC-3’; Fly_GAPDH1_R: 5’-GGG CCG AGA TGA TGA CCT TCT T-3’
Take 3 μl RT-PCR product to run 1.5% agarose gel. QC is passed if positive control (Mme_F + fly_GAPDH1_R) shows specific band, experimental control (Mme_F + Mme_R) shows strong smear and single primer amplification (Mme_F only or Mme_R only) shows no or weak smear.
Amplification of cDNA by low-cycle PCR
Timing: 1 h
- Prepare the following in a UV treated PCR tube: cDNA from step 19 16 μl
5x HF buffer (NEB) 10 μl
10 μM Mme_F (IDT) 2.5 μl
10 μM Mme_R (IDT) 2.5 μl
10 mM dNTP, each (Bioline) 1 μl
Phusion HF DNA polymerase (NEB) 0.5 μl
Nuclease-free water 17.5 μl
Total reaction volume 50 μl
Perform the thermal cycling: 98 °C for 30 sec; 5 cycles of 98 °C for 10 sec, 67 °C for 10 se and 72 °C for 90 sec; 72 °C for 10 min. If starting with less amount of RNA, more cycles can be added (up to 15 cycles).
Exo I digestion to remove excess primers
Timing: 1.5 h
- Add 5 μl Exonuclease I (Exo I) to the 50 μl PCR reaction. Incubate at 37 °C for 45 min, 80 °C for 20 min, and hold the reaction at 10 °C. Purify the samples with ZYMO Clean & Concentrator-5 kit. Elute with 10 μl nuclease-free water. TIP: To avoid airborne DNA contamination, use fresh binding buffer and wash buffer of the ZYMO kit. Or filter the buffer with 0.2 μm filter unit and treated with UV.
Circularization of PCR product
Timing: 3 h
- Combine the following in a UV treated PCR tube: Purified PCR product from step 21 10 μl
10x Ampligase buffer (Epicentre) 3 μl
OptiKinase (USB) 1.5 μl
100 mM ATP (VWR) 1.5 μl
100 mM DTT (Invitrogen) 0.3 μl
Nuclease-free water 11.3 μl
Reaction volume at this point 27.6 μl
Incubate the reaction at 37 °C for 30 min, then 95 °C for 2 min to heat inactivate OptiKinase. Hold the reaction at 4 °C.
Add the following two components in PCR hood to avoid potential contamination:
10 μM TSS_Col3_short (IDT) 0.9 μl
Ampligase (Epicentre) 1.5 μl
Total reaction volume 30 μl
Mix the reaction and perform thermal cycling as following: 5 cycles (30 sec at 95 °C, 2 min at 68 °C, 1 min at 55 °C, 5 min at 60 °C); 5 cycles (30 sec at 95 °C, 2 min at 65 °C, 1 min at 55 °C, 5 min at 60 °C). Hold the reaction at 10 °C.
Sequence of TSS_Col3_short: 5’-GCC GTC TTC AGC CGC CTCA AGC TTC TAA CGA TGT ACG-3’
Critical step: Any circular DNA contamination in this step will be amplified in the following RCA step and should be avoided2. Use Ampligase buffer, 100mM ATP and 100mM DTT which were aliquoted and kept at -20°C. UV treats tubes and nuclease-free water to remove potential DNA contaminations.
Exonuclease digestion
Timing: 1.5 h
Add 3 μl Exo I (NEB) and 0.6 μl Exo III (NEB) to the 30 μl circularization reaction. Incubate 45 min at 37 °C, 20 min at 80 °C, and hold at 10 °C. This step eliminates DNA fragments in linear form, and only single-stranded circular DNA remain after the digestion. Directly use the reaction for rolling circle amplification (RCA).
Rolling circle amplification (RCA)
Timing: 17 h
- Combine the following in four UV treated PCR tubes: Circular DNA from previous step 2 μl
25 mM dNTP, each (Epicentre) 0.8 μl
10 mg/ml BSA (NEB) 0.4 μl
10 x Phi29 buffer (NEB) 2 μl
100 μM N6T2 (IDT) 2 μl
Phi29 high fidelity DNA polymerase (NEB) 1 μl
DMSO, UV-treated (Stratagene) 2 μl
H2O (Ambion, UV-treated) 9.8 μl
Total reaction volume 20 μl
TIP: Before adding Phi29 DNA polymerase, heat the reaction at 80 °C for 2 min and snap cool down on ice for 2 min to reduce bias of random primer binding. N6T2: 5’-NNNNNN-3’; * = phosphothiol.
Tips: Perform multiple reactions to reduce amplification bias. We typically prepare four reactions for each sample.
Mix the reactions by pipetting and incubate them as following: 10 °C for 10 min, 28 °C for 16 h, 65 °C for 10 min, and hold at 10 °C. Combine the four tubes of same sample after RCA.
Quality control step 2
This QC step is to determine whether the circular DNAs are specifically amplified using a built-in XhoI site in the 5’ adaptor. This step also can help to estimate the distribution of the circular molecules.
Timing: 1.5 h
- QC by XhoI digestion. Prepare the following reaction: RCA product 2 μl
10 x buffer 2 (NEB) 2 μl
100 x BSA (NEB) 0.2 μl
XhoI (NEB) 1 μl
H2O 14.8 μl
Total reaction volume 20 μl
Incubate the reaction at 37 °C for 1 h. Take 10 μl reaction to run in a 1.5% agarose gel. Results passed QC: Majority of the RCA products were digested by XhoI and generate smear ranging from 200 bp to 1000 bp and peaked at ~400 bp.
Precipitation of RCA products
Timing: 1.5 h
For each sample, combine 4 tubes of RCA products together. Add 120 μl nuclease-free water to bring a total volume of 200 μl. Extract once with phenol/chloroform then with chloroform followed by ethanol precipitation. Qualify the RCA products by Nanodrop spectrophotometer. The typical yield is around 60 μg.
Generation of ditag by MmeI digestion
Timing: 2.5 h
- Prepare the following reaction in a 1.7 ml tube: RCA product from previous step 16 μg
10x buffer 4 (NEB) 18 μl
SAM, dilute to 500 μM using 1x NEB buffer 4 20 μl
MmeI, 2U/μl (NEB) 10 μl
Nuclease-free water add to 200 μl
Total reaction volume 200 μl
TIP: The original concentration of SAM is 32 mM. Use 1x buffer 4 (NEB) to dilute SAM.
Incubate the reaction at 37 °C for 30 min. MmeI digestion enables the release of small fragment (94bp) which containing ditag3. Concentrate the reaction to 12 μl with two DNA clean & concentrator-5 columns (ZYMO). Load 6 μl of purified DNA to one lane (one sample has two lanes) in 6% acrylamide gel. We use 18 x 16 cm acylamide gel to obtain better separation of MmeI digested product (94 bp) from background. Run the vertical gel at 200 V for 1.5 h. Compare the intensity of bands with the DNA standard (100 bp band of Invitrogen’s 10 bp DNA ladder), we estimate that the desired DNA fragment is ~7.2 pmol.
Purification of MmeI digested small fragment
Timing: overnight
Use a new Razor Blade (VWR) to cut the desired band (~94 bp) which contains both TSS sequence and 3’ downstream tag. Elute each gel slice with 400 μl gel elution buffer (0.1% SDS, 0.2 NaCl, 10 mM MgOAc2). Rotate at least 6 h at room temperature or overnight at 4 °C. Phenol/chloroform extraction followed by ethanol precipitation. Resuspend the sample in 20 μl nuclease-free water.
Ligation with paired-end linkers
Timing: overnight
- Combine the following in a PCR tube: Purified DNA from previous step 20 μl
12.5 μM PE2_AN2 1.5 μl
12.5 μM PE2_BN2 1.5 μl
T4 DNA ligase buffer (NEB) 5 μl
40% PEG8000 (filtered and autoclaved) 20 μl
T4 DNA ligase, 2000U/ul (NEB) 2 μl
Total reaction volume 50 μl
Incubate the reaction overnight at 16 °C.
TIP: Mix the reaction for at least 20 times to ensure the even distribution of PEG8000.
Linker sequences:
PE2_AN2:
5’-ACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNN-3’
3’-TGGCTCTAGATGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGA-p-5’
PE2_BN2:
5’-p-AGATCGGAAGAGCGGTTCAGCAGGAATGCCGAGACCGATCT-3’
3’-NNTCTAGCCTTCTCGCCAAGTCGTCCTTACGGCTCTGGCTAGA-5’
‘p’ denotes phosphate group; ‘N’ represents any nucleotide.
Phenol/chloroform extraction followed by concentrate with ZYMO Clean & Concentrator-5 kit. Elute the ligation product with 10 μl nuclease-free water.
Final library amplification by PCR
Timing: 1.5 h
- Prepare the following in a PCR tube: Linker ligated DNA from previous step 10 μl
5x HF buffer (NEB) 10 μl
1 μM PE2_AFN2_short (IDT) 2.5 μl
10 μM PE2_A_short (IDT) 2.5 μl
1 μM PE2_BRN2_short (IDT) 2.5 μl
10 μM PE2_B_short (IDT) 2.5 μl
10 mM dNTP, each (Bioline) 1 μl
Phusion HF DNA polymerase (NEB) 0.5 μl
Nuclease-free water 18.5 μl
Total reaction volume 50 μl
Sequence of primers:
PE2_AFN2_short: 5’-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACA-3’
PE2_BRN2_short: 5’-CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCT-3’
PE2_A_short: 5’-AATGATACGGCGACCACCGAGA-3’
PE2_B_short: 5’-CAAGCAGAAGACGGCATACGAGA-3’
Run the following PCR program: incubate for 30 sec at 98 °C, followed by a 2 cycles of (10 sec and 98 °C, 10 sec at 66 °C, 30 sec at 72°C) and 10 cycles of (10 sec and 98 °C, 10 sec at 69 °C, 30 sec at 72°C), additional extension for 10 min at 72 °C and then hold at 10 °C.
Quality control step 3
Timing: 0.5 h
- Take 3 μl of PCR product to run a 1.5% agarose gel. One should see a specific band of 213 bp. If very strong non-specific amplification occurs, perform size selection (~180 bp) after linker ligation. TIP: A library with good quality should have a relatively strong band (~213 bp) compared to the primer dimers.
Size selection in an acrylamide gel
Timing: overnight
- Use ZYMO DNA clean & concentrator-5 kit to purify the remaining 47 μl PCR product and elute with 6 μl nuclease-free water. Load the purified sample to an 8% acrylamide gel and run at 1 w for 1 hr. Stain the acrylamide gel with EtBr for 2 min and cut the ~213 bp band under UV transilluminator. Elute the gel slice with 400 μl gel elution buffer (0.1% SDS, 0.3M NaOAc, pH5.2) and rotate at room temperature for at least 6 h or at 4 °C overnight. Purify the final library with ZYMO DNA clean & concentrator-5 kit. Load multiple times for mixture more than 700 μl. Elute with 30 μl nuclease-free water. Quantify the final library by Qubit fluoromerter according to manufacturer’s protocol. The concentration of our sample (fly 0-24h embryo) is 1.3 ng/μl. TIP: This size selection step is to remove non-specific amplification. It helps to increase the quality of paired-end library.
Quality control step 4
We use Sanger sequencing to evaluate the quality of the final TSS library.
Timing: two days
Use 5 μl out of 30 μl final library for the QC by Sanger sequencing. We obtained 20 qualified clones; 18 of the clones could be mapped to annotated Drosophila TSSs (± 250nt).
Paired-end sequencing by Illumina GAIIx
Timing: 3~4 days
- The final library passed QC was subjected to paired-end sequencing by Illumina Genome Analyzer IIx.
The whole procedure takes ~7 days.
Reverse transcription with random primer
Step 13 and 14
The ratio of primer to template is critical to the distance distribution of 5’ TSS tag and 3’ downstream tag.
Circularization of PCR product
Step 22
More efficient of the circularization step is, the higher the quality of the final PEAT library.
Generation of ditag by MmeI digestion
Step 29 and 30
RCA product and enzyme should be mixed at indicated ratio to ensure efficient digestion. More recovery of desired band (~94bp) reduces the bias of the final PEAT library.
Purification of MmeI digested small fragment
Step 31
Use large vertical gel to separate the desired band (94bp); this will increase the specificity of the final PEAT library. A small fraction of undigested RCA product will be introduced in the 94bp band during gel cut, thus large gel enables more precious separation.
Total RNA is not of high quality:
Use RNase-free reagent and RNase-free water to isolate total RNA. All microtubes should be autoclaved and UV treated. Use RNaseZap RNase Decontamination Solution (Ambion) to wipe benchtop and all pipettes.
RCA is not efficient:
Multiple reasons may cause this problem. One possibility is less circular DNA is generated during circularization step. Always do quality control step 2 to make sure cDNA can be specifically amplified by primer pair Mme_F and Mme_R. Another reason is Phi29 DNA polymerase might be inactivated by UV treatment if the temperature gets too high during treatment. To solve this, make sure the PCR cap which contains enzyme meets cooled water in the well of the PCR cooler during the treatment. After adding water to the well of the PCR cooler, wait for at least 1 min to let the water to cool down to ~0 °C.
Sanger sequencing revealed high percentage of clones without built-in linkers:
The built-in linkers are used to distinguish if the 20 nt tag is derived from the TSS side or from the 3’ downstream region. Unsuccessful separation of the 94 bp band from background is the main reason. To resolve this problem, always remember to use large acrylamide gel to better separate the intended band from background. In addition, cut the ~94 bp band as tight as possible. All these efforts will reduce the background to less than 5%.
Deep sequencing results revealed that 90% of the paired reads have built-in linkers. Of those paired reads, 76% were mapped to a unique location within Release 5 of the fly genome. An additional 10% of pairs mapped to multiple genomic locations. The median distance between the 5’ and 3’ reads at the transcript level was 279 nt. 81.5% of genes currently annotated by FlyBase (v5.14) were represented by at least one read-pair.
- Collart, M.A. & Oliviero, S. Preparation of yeast RNA. Current protocols in molecular biology / edited by Frederick M. Ausubel et al., Chapter 13, Unit13 12 (2001).
- Zhang, K. et al. Sequencing genomes from single cells by polymerase cloning. Nature biotechnology 24, 680-686 (2006).
- Shendure, J. et al. Accurate multiplex polony sequencing of an evolved bacterial genome. Science 309, 1728-1732 (2005).
We are grateful to David MacAlpine and Sara Powell (Duke University Medical Center) for their help in collecting fly embryos. We thank Bin Xie (Virginia Commonwealth University) for his effort in performing the Illumina paired-end sequencing. We are indebted to Dr. Yongde Bao (University of Virginia) for his effort in optimizing the paired-end sequencing procedure.
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Associated Publications
A paired-end sequencing strategy to map the complex landscape of transcription initiation
by Ting Ni, David L Corcoran, Elizabeth A Rach, +5
Nature Methods (22 May, 2012)
Method Article
Paired-end analysis of transcriptional start sites
Ting Ni
Duke University
Corresponding Author
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We describe an improved deep sequencing strategy, paired-end analysis of transcriptional start sites (PEAT), to globally characterize transcriptional start sites (TSSs) in a previous study [1]. It was used to explore the landscape of transcription initiation in Drosophila melanogaster embryo. Here we present a detailed protocol, with minor modifications, can be broadly applied to investigate capped transcripts in other eukaryotes, from budding yeast to rat and human cell lines.
Figure 1
Figure 2
Figure 3
TRIzol reagent (Invitrogen, cat. no. 15596-018)
Nuclease-free Water (Ambion, cat. no. AM9932 )
RNaseZap RNase Decontamination Solution (Ambion, cat. no. AM9780)
Phenol:Chloroform:Isoamyl Alcohol (25:24:1), pH8.0 (Sigma, cat. no. P2069)
Chloroform (VWR, cat. no. MK444010)
Glacial acetic acid (VWR, cat. no. 87003-238)
2-Propanol (Sigma, cat. no. I9516)
Absolute ethanol (VWR, cat. no. 200004-482)
Formaldehyde solution (Sigma, cat. no. F8775)
Trizma hydrochloride (Sigma, cat. no. T5941)
Trizma base (Sigma, cat. no. T6066)
Sodium chloride (Sigma, cat. no. S3014)
Sodium acetate (Sigma, cat. no. S7670)
EDTA (Sigma, cat. no. E5134)
SDS (Sigma, cat. no. L4390)
Boric acid (Sigma, cat. no. B7901)
Urea (Sigma, cat. no. U0631)
Lithium chloride (Sigma, cat. no. L9650)
Lithium dodecyl sulfate (Sigma, cat. no. L9781)
Triton X-100 (Sigma, cat. no. T8787)
DMSO (Sigma, cat. no. D8418)
Agarose (Bio-Rad, cat. no. 161-3102EDU)
Ethidium Bromide Solution (Bio-Rad, cat. no. 161-0433)
40% acrylamide and bis-acrylamide solution, 19:1 (Bio-Rad, cat. no. 161-0144)
Ammonium Persulfate (Bio-Rad, cat. no. 161-0700)
TEMED (Bio-Rad, cat. no. 161-0800)
RNeasy Mini kit (Qiagen, cat. no. 74104)
RNeasy MinElute Cleanup Kit (Qiagen, cat. no. 74204)
RNase-Free DNase Set (Qiagen, cat. no. 79254)
Dynabeads Oligo(dT)25 (Invitrogen, cat. no. 610-02)
SuperScript III Reverse Transcriptase (Invitrogen, cat. no. 18080-093)
Bacterial Alkaline Phosphatase, E.coli C75 (Takara, cat. no. TAK 2120B)
Ampligase Thermostable DNA Ligase (Epicentre, cat. no. A32750)
Tobacco Acid Pyrophosphatase (Epicentre, cat. no. T19100)
T4 RNA Ligase 1 (NEB, cat. no. M0204L)
Phusion High-Fidelity DNA Polymerase (NEB, cat. no. F-530L)
Taq DNA Polymerase (NEB, cat. no. M0273L)
Exo I nuclease (NEB, cat. no. M0265S)
Exo III nuclease (NEB, cat. no. M0206S)
Phi29 DNA polymerase (NEB, cat. no. M0269L)
MmeI (NEB, cat. no. R0637L)
XhoI (NEB, cat. no. R0146M)
RNasin ribonuclease inhibitor (Promega, cat. no. N2515)
dNTP Mix (Bioline, cat. no. BIO-39029)
HyperLadde I DNA ladder (Bioline, cat. no. BIO-33026)
HyperLadde IV DNA ladder (Bioline, cat. no. BIO-33030)
10 bp DNA Ladder (Invitrogen, cat. no. 10821-015)
Ribonuclease H (Invitrogen, cat. no. 18021-014)
OptiKinase (USB, cat. no. 78334Y)
Actinomycin D (USB, cat. no. 10415)
GlycoBlue (Ambion, cat. no. AM9516)
ZYMO DNA clean & concentrator-5 kit (ZYMO, cat. no. D4014)
PEG 8000 (VWR, cat. no. PAV3011)
Biotin (Long arm) Hydrazide (VWR, cat. no. 101098-478)
ATP solution (VWR, cat. no. 100216-332)
pGEM-T Easy Vector System II (Promega, cat. no. A1380)
1.7 ml microcentrifuge tube (Denville Scientific, cat. no. C2170)
PCR tube strips and cap strips (Axygen Scientific, cat. no. PCR-0208CPC)
Barrier tips, 10 ul (Denville Scientific, cat. no. P1096-FR)
Barrier tips, 20 ul (Denville Scientific, cat. no. P1121)
Barrier tips, 200 ul (Denville Scientific, cat. no. P1122)
Barrier tips, 1250 ul (Denville Scientific, cat. no. P1126)
Microcentrifuge (Eppendorf Model 5424)
Microcentrifuge (Eppendorf Model 5415R)
Pellet pestles blue polypropylene (Sigma, cat. no. Z359947)
Razor Blades (VWR, cat. no. 55411-050)
NanoDrop 1000 Spectrophotometer (Thermo Scientific, cat. no. SID-10135606)
Standard PCR machine (e.g., PTC-200 Thermo Cycler, MJ Research)
Qubit Fluorometer (Invitrogen, cat. no. Q32857)
PCR-Cooler (Eppendorf, cat. no. 022510509)
UV Stratalinker 2400 (Stratagene)
AC600 PCR workstation (AirClean system)
Dynal MPC-T4 Magnet (Invitrogen)
Tube Rotator (VWR, cat. no. 13916-822)
Mini-vertical gel electrophoresis unit (Hoefer, cat. no. SE250)
Sturdier vertical electrophoresis unit (Hoefer, cat. no. SE410)
Standard heating block (e.g., Analog Dry Block Heater, VWR)
Standard water bath (e.g., Analog and Digital Unstirred Water Baths, VWR)
Gel imaging systems (e.g., AlphaImager, Alpha Innotech)
Standard power supply (e.g., PowerPac HC Power Supply, Bio-Rad)
Preparation of RNA samples
Timing: 1h
The quality of the starting RNA samples is critical for the success of the PEAT strategy. We recommend TRIzol reagent to isolate total RNA from higher eukaryotes (both animal and plant). For yeast cells, hot-phenol approach1 tends to give the most reproducible results. To avoid potential biases that may incur during library construction, it is preferable to start with ~150 μg total RNA if the sample is easy to be obtained. For low-abundance samples, it is possible to reduce the amount to 10 μg. In this protocol, we use Drosophila embryos to demonstrate the PEAT strategy. One can adapt the protocol to construct PEAT libraries for other species as well
RNA cleanup
Timing: 1 h
RNeasy Mini kit (QIAGEN) is used to remove genomic DNA contamination in RNA preparation. On-column DNase I digestion step is included according to the manufacturer’s protocol Starting with ~150 μg total RNA, the final product is eluted with 80 μl nuclease-free water.
(Optional Step) The integrity of the purified RNA is checked with1.2% formaldehyde agarose gel (FA gel). Alternatively, Agilent RNA Nano Labchip can be used to determine the RNA quality. Proceed to the next step if the RNA samples pass quality control.
PolyA+ RNA selection with Dynabeads (dT)25
Timing: 3 h
Perform two rounds of polyA+ selection with Dynabeads (dT)25 according to manufacturer’s protocol. Use all of the purified total RNAs from the previous step and elute the polyA+ RNA with 30 μl elution buffer (10mM Tris-HCl, pH7.5).
Lithium salt resulting from polyA+ selection step might affect downstream enzymatic activity. RNeasy MinRlute kit is therefore used to remove residual lithium by following the manufacturer’s protocol. We recommend to elute the column twice, each with 40 μl nuclease-free water. The resulting products are then quantified with a nanodrop spectrophotometer. From 150 μg total RNA, the typical yield of purified polyA+ RNA is 2-3 μg.
Bacterial Alkaline Phosphatase (BAP) treatment
Timing: 2.5 h
- Preparing the following reaction in an RNase-free 1.7ml tube: RNA from step 5 80 μl
BAP enzyme (Takara) 6 μl
10x BAP buffer (Takara) 10 μl
RNasin ribonuclease inhibitor (Promega) 2.5 μl
Nuclease-free water 1.5 μl
Total reaction volume 100 μl
- Incubate the reaction at 37 °C for 45 min. Add 100 μl nuclease-free water to the reaction. Extract once with 200 μl phenol/chloroform and then with 200 μl chloroform. Perform ethanol precipitation by adding 20 μl 3 M NaOAc (pH 5.2), 2 μl GlycoBlue and 500 μl ethanol. Wash the pellet once with 70% ethanol, followed by adding 85.5 μl nuclease-free water to dissolve RNA. Critical step: Completely removal of the phosphate group on the uncapped RNA molecules before TAP treatment is critical for reducing background noise.
Tobacco Acid Pyrophosphatase (TAP) treatment
Timing: 2.5 h
- Preparing the following reaction in a RNase-free 1.7 ml tube: BAP treated RNA from step 7 85.5 μl
10x TAP buffer (Epicentre) 10 μl
TAP enzyme (Epicentre) 2 μl
RNasin ribonuclease inhibitor (Promega) 2.5 μl
Total reaction volume 100 μl
Incubate the reaction at 37 °C for 1 h. Add 100 μl nuclease-free water to the reaction. Extract once with 200 μl phenol/chloroform and then with 200 μl chloroform. Perform ethanol precipitation by adding 20 μl 3 M NaOAc (pH 5.2), 2 μl GlycoBlue and 500 μl ethanol. Wash the pellet once with 70% ethanol. Dissolve the BAP and TAP treated RNA in 13 μl nuclease-free water.
Ligation with 5’ chimeric linker
Timing: 3 h (or overnight)
- Combing the following in a 1.7ml RNase-free tube: BAT/TAP treated polyA+ RNA from step 9 13 μl
30 μM PAGE purified Mme_CAP_RDhyb (IDT) 2 μl
10x T4 RNA ligase buffer (NEB) 10 μl
40% PEG8000 (0.2 μM filtered and autoclaved) 62.5 μl
RNasin ribonuclease inhibitor (Promega) 2.5 μl
T4 RNA ligase1 (NEB) 10 μl
Total reaction volume 100 μl
Critical step: The ratio of linker:RNA should be larger than 10:1 to ensure efficient ligation. The sequence of Mme_CAP_RDhyb: 5’-CTC AAG CTT CTA ACG ATG TAC GCT CG rArGrUrCrCrArArC-3’, ‘r’ denotes ribonucleotide.
Mix the reaction well by pipetting and incubate at room temperature for at least 3 h (or overnight). Add 100 μl nuclease-free water to the reaction and extract once with 200 μl phenol/chloroform and a second time with 200 μl chloroform.
Removal of excess linkers
Timing: 0.5 h
The supernatant from Step 11 is further purified with RNeasy MinElute kit (QIAGEN) to remove excess linkers by following the manufacturer’s protocol. Elute linker-ligated RNA with 20 μl nuclease-free water.
Reverse transcription with random primer
Timing: 2 h
- Prepare the reaction in a PCR tube: RNA sample from previous step 20 μl
10mM dNTP, each (Bioline) 2 μl
10uM Mme_RTN6 (IDT) 2 μl
Incubate the reaction for 5 min at 65 °C, then chill on ice for ≥ 1 min. Add the following:
5x first strand buffer (Invitrogen) 8 μl
0.1M DTT (Invitrogen) 2 μl
RNasin ribonuclease inhibitor (Promega) 2 μl
20x Actinomycin D (USB) 2 μl
SuperScript III reverse transcriptase (Invitrogen) 2 μl
Total reaction volume 40 μl
The sequence of Mme_RTN6: 5’-GCG GCT GAA GAC GGC CTA TCC GAC NNN NNN-3’
Mix and incubate the reaction at 25 °C for 5 min, 50 °C for 1 h, 70 °C 15 min. Hold at 10 °C.
- Add 1 μl RNase H (2 Units, Invitrogen) to the reaction and incubate at 37 °C for 20 min. This treatment helps to remove RNA strand from RNA:DNA hybrid. Use ZYMO DNA clean & concentrator-5 kit to purify the samples following manufacturer’s protocol. Elute with 20 μl nuclease-free water. TIP: Add at least 7-fold of the binding buffer to efficiently recover single-stranded cDNA.
Quality control step 1
RT-PCR is used to evaluate the efficiency of linker ligation.
Timing: 2 h
- Prepare the following in a PCR tube: Purified cDNA from step 15 0.25 μl
5x HF buffer (NEB) 4 μl
10 μM Mme_F (IDT) 1 μl
10 μM Mme_R or fly_GAPDH1_R (IDT) 1ul
10 mM dNTP, each (Bioline) 0.4 μl
Phusion high fidelity DNA polymerase (NEB) 0.2 μl
Nuclease-free water 13.15 μl
Total reaction volume 20 μl
- Perform the thermal cycling below: 98 °C for 30 sec; 30 cycles of 98 °C for 10 sec, 67 °C for 10 sec and 72 °C for 90 sec; 72 °C for 10 min. Sequences of PCR primers: Mme_F: 5’-CTC AAG CTT CTA ACG ATG TAC GCT CGA-3’; Mme_R: 5’-GCG GCT GAA GAC GGC CTA TCC-3’; Fly_GAPDH1_R: 5’-GGG CCG AGA TGA TGA CCT TCT T-3’
Take 3 μl RT-PCR product to run 1.5% agarose gel. QC is passed if positive control (Mme_F + fly_GAPDH1_R) shows specific band, experimental control (Mme_F + Mme_R) shows strong smear and single primer amplification (Mme_F only or Mme_R only) shows no or weak smear.
Amplification of cDNA by low-cycle PCR
Timing: 1 h
- Prepare the following in a UV treated PCR tube: cDNA from step 19 16 μl
5x HF buffer (NEB) 10 μl
10 μM Mme_F (IDT) 2.5 μl
10 μM Mme_R (IDT) 2.5 μl
10 mM dNTP, each (Bioline) 1 μl
Phusion HF DNA polymerase (NEB) 0.5 μl
Nuclease-free water 17.5 μl
Total reaction volume 50 μl
Perform the thermal cycling: 98 °C for 30 sec; 5 cycles of 98 °C for 10 sec, 67 °C for 10 se and 72 °C for 90 sec; 72 °C for 10 min. If starting with less amount of RNA, more cycles can be added (up to 15 cycles).
Exo I digestion to remove excess primers
Timing: 1.5 h
- Add 5 μl Exonuclease I (Exo I) to the 50 μl PCR reaction. Incubate at 37 °C for 45 min, 80 °C for 20 min, and hold the reaction at 10 °C. Purify the samples with ZYMO Clean & Concentrator-5 kit. Elute with 10 μl nuclease-free water. TIP: To avoid airborne DNA contamination, use fresh binding buffer and wash buffer of the ZYMO kit. Or filter the buffer with 0.2 μm filter unit and treated with UV.
Circularization of PCR product
Timing: 3 h
- Combine the following in a UV treated PCR tube: Purified PCR product from step 21 10 μl
10x Ampligase buffer (Epicentre) 3 μl
OptiKinase (USB) 1.5 μl
100 mM ATP (VWR) 1.5 μl
100 mM DTT (Invitrogen) 0.3 μl
Nuclease-free water 11.3 μl
Reaction volume at this point 27.6 μl
Incubate the reaction at 37 °C for 30 min, then 95 °C for 2 min to heat inactivate OptiKinase. Hold the reaction at 4 °C.
Add the following two components in PCR hood to avoid potential contamination:
10 μM TSS_Col3_short (IDT) 0.9 μl
Ampligase (Epicentre) 1.5 μl
Total reaction volume 30 μl
Mix the reaction and perform thermal cycling as following: 5 cycles (30 sec at 95 °C, 2 min at 68 °C, 1 min at 55 °C, 5 min at 60 °C); 5 cycles (30 sec at 95 °C, 2 min at 65 °C, 1 min at 55 °C, 5 min at 60 °C). Hold the reaction at 10 °C.
Sequence of TSS_Col3_short: 5’-GCC GTC TTC AGC CGC CTCA AGC TTC TAA CGA TGT ACG-3’
Critical step: Any circular DNA contamination in this step will be amplified in the following RCA step and should be avoided2. Use Ampligase buffer, 100mM ATP and 100mM DTT which were aliquoted and kept at -20°C. UV treats tubes and nuclease-free water to remove potential DNA contaminations.
Exonuclease digestion
Timing: 1.5 h
Add 3 μl Exo I (NEB) and 0.6 μl Exo III (NEB) to the 30 μl circularization reaction. Incubate 45 min at 37 °C, 20 min at 80 °C, and hold at 10 °C. This step eliminates DNA fragments in linear form, and only single-stranded circular DNA remain after the digestion. Directly use the reaction for rolling circle amplification (RCA).
Rolling circle amplification (RCA)
Timing: 17 h
- Combine the following in four UV treated PCR tubes: Circular DNA from previous step 2 μl
25 mM dNTP, each (Epicentre) 0.8 μl
10 mg/ml BSA (NEB) 0.4 μl
10 x Phi29 buffer (NEB) 2 μl
100 μM N6T2 (IDT) 2 μl
Phi29 high fidelity DNA polymerase (NEB) 1 μl
DMSO, UV-treated (Stratagene) 2 μl
H2O (Ambion, UV-treated) 9.8 μl
Total reaction volume 20 μl
TIP: Before adding Phi29 DNA polymerase, heat the reaction at 80 °C for 2 min and snap cool down on ice for 2 min to reduce bias of random primer binding. N6T2: 5’-NNNNNN-3’; * = phosphothiol.
Tips: Perform multiple reactions to reduce amplification bias. We typically prepare four reactions for each sample.
Mix the reactions by pipetting and incubate them as following: 10 °C for 10 min, 28 °C for 16 h, 65 °C for 10 min, and hold at 10 °C. Combine the four tubes of same sample after RCA.
Quality control step 2
This QC step is to determine whether the circular DNAs are specifically amplified using a built-in XhoI site in the 5’ adaptor. This step also can help to estimate the distribution of the circular molecules.
Timing: 1.5 h
- QC by XhoI digestion. Prepare the following reaction: RCA product 2 μl
10 x buffer 2 (NEB) 2 μl
100 x BSA (NEB) 0.2 μl
XhoI (NEB) 1 μl
H2O 14.8 μl
Total reaction volume 20 μl
Incubate the reaction at 37 °C for 1 h. Take 10 μl reaction to run in a 1.5% agarose gel. Results passed QC: Majority of the RCA products were digested by XhoI and generate smear ranging from 200 bp to 1000 bp and peaked at ~400 bp.
Precipitation of RCA products
Timing: 1.5 h
For each sample, combine 4 tubes of RCA products together. Add 120 μl nuclease-free water to bring a total volume of 200 μl. Extract once with phenol/chloroform then with chloroform followed by ethanol precipitation. Qualify the RCA products by Nanodrop spectrophotometer. The typical yield is around 60 μg.
Generation of ditag by MmeI digestion
Timing: 2.5 h
- Prepare the following reaction in a 1.7 ml tube: RCA product from previous step 16 μg
10x buffer 4 (NEB) 18 μl
SAM, dilute to 500 μM using 1x NEB buffer 4 20 μl
MmeI, 2U/μl (NEB) 10 μl
Nuclease-free water add to 200 μl
Total reaction volume 200 μl
TIP: The original concentration of SAM is 32 mM. Use 1x buffer 4 (NEB) to dilute SAM.
Incubate the reaction at 37 °C for 30 min. MmeI digestion enables the release of small fragment (94bp) which containing ditag3. Concentrate the reaction to 12 μl with two DNA clean & concentrator-5 columns (ZYMO). Load 6 μl of purified DNA to one lane (one sample has two lanes) in 6% acrylamide gel. We use 18 x 16 cm acylamide gel to obtain better separation of MmeI digested product (94 bp) from background. Run the vertical gel at 200 V for 1.5 h. Compare the intensity of bands with the DNA standard (100 bp band of Invitrogen’s 10 bp DNA ladder), we estimate that the desired DNA fragment is ~7.2 pmol.
Purification of MmeI digested small fragment
Timing: overnight
Use a new Razor Blade (VWR) to cut the desired band (~94 bp) which contains both TSS sequence and 3’ downstream tag. Elute each gel slice with 400 μl gel elution buffer (0.1% SDS, 0.2 NaCl, 10 mM MgOAc2). Rotate at least 6 h at room temperature or overnight at 4 °C. Phenol/chloroform extraction followed by ethanol precipitation. Resuspend the sample in 20 μl nuclease-free water.
Ligation with paired-end linkers
Timing: overnight
- Combine the following in a PCR tube: Purified DNA from previous step 20 μl
12.5 μM PE2_AN2 1.5 μl
12.5 μM PE2_BN2 1.5 μl
T4 DNA ligase buffer (NEB) 5 μl
40% PEG8000 (filtered and autoclaved) 20 μl
T4 DNA ligase, 2000U/ul (NEB) 2 μl
Total reaction volume 50 μl
Incubate the reaction overnight at 16 °C.
TIP: Mix the reaction for at least 20 times to ensure the even distribution of PEG8000.
Linker sequences:
PE2_AN2:
5’-ACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNN-3’
3’-TGGCTCTAGATGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGA-p-5’
PE2_BN2:
5’-p-AGATCGGAAGAGCGGTTCAGCAGGAATGCCGAGACCGATCT-3’
3’-NNTCTAGCCTTCTCGCCAAGTCGTCCTTACGGCTCTGGCTAGA-5’
‘p’ denotes phosphate group; ‘N’ represents any nucleotide.
Phenol/chloroform extraction followed by concentrate with ZYMO Clean & Concentrator-5 kit. Elute the ligation product with 10 μl nuclease-free water.
Final library amplification by PCR
Timing: 1.5 h
- Prepare the following in a PCR tube: Linker ligated DNA from previous step 10 μl
5x HF buffer (NEB) 10 μl
1 μM PE2_AFN2_short (IDT) 2.5 μl
10 μM PE2_A_short (IDT) 2.5 μl
1 μM PE2_BRN2_short (IDT) 2.5 μl
10 μM PE2_B_short (IDT) 2.5 μl
10 mM dNTP, each (Bioline) 1 μl
Phusion HF DNA polymerase (NEB) 0.5 μl
Nuclease-free water 18.5 μl
Total reaction volume 50 μl
Sequence of primers:
PE2_AFN2_short: 5’-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACA-3’
PE2_BRN2_short: 5’-CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCT-3’
PE2_A_short: 5’-AATGATACGGCGACCACCGAGA-3’
PE2_B_short: 5’-CAAGCAGAAGACGGCATACGAGA-3’
Run the following PCR program: incubate for 30 sec at 98 °C, followed by a 2 cycles of (10 sec and 98 °C, 10 sec at 66 °C, 30 sec at 72°C) and 10 cycles of (10 sec and 98 °C, 10 sec at 69 °C, 30 sec at 72°C), additional extension for 10 min at 72 °C and then hold at 10 °C.
Quality control step 3
Timing: 0.5 h
- Take 3 μl of PCR product to run a 1.5% agarose gel. One should see a specific band of 213 bp. If very strong non-specific amplification occurs, perform size selection (~180 bp) after linker ligation. TIP: A library with good quality should have a relatively strong band (~213 bp) compared to the primer dimers.
Size selection in an acrylamide gel
Timing: overnight
- Use ZYMO DNA clean & concentrator-5 kit to purify the remaining 47 μl PCR product and elute with 6 μl nuclease-free water. Load the purified sample to an 8% acrylamide gel and run at 1 w for 1 hr. Stain the acrylamide gel with EtBr for 2 min and cut the ~213 bp band under UV transilluminator. Elute the gel slice with 400 μl gel elution buffer (0.1% SDS, 0.3M NaOAc, pH5.2) and rotate at room temperature for at least 6 h or at 4 °C overnight. Purify the final library with ZYMO DNA clean & concentrator-5 kit. Load multiple times for mixture more than 700 μl. Elute with 30 μl nuclease-free water. Quantify the final library by Qubit fluoromerter according to manufacturer’s protocol. The concentration of our sample (fly 0-24h embryo) is 1.3 ng/μl. TIP: This size selection step is to remove non-specific amplification. It helps to increase the quality of paired-end library.
Quality control step 4
We use Sanger sequencing to evaluate the quality of the final TSS library.
Timing: two days
Use 5 μl out of 30 μl final library for the QC by Sanger sequencing. We obtained 20 qualified clones; 18 of the clones could be mapped to annotated Drosophila TSSs (± 250nt).
Paired-end sequencing by Illumina GAIIx
Timing: 3~4 days
- The final library passed QC was subjected to paired-end sequencing by Illumina Genome Analyzer IIx.
The whole procedure takes ~7 days.
Reverse transcription with random primer
Step 13 and 14
The ratio of primer to template is critical to the distance distribution of 5’ TSS tag and 3’ downstream tag.
Circularization of PCR product
Step 22
More efficient of the circularization step is, the higher the quality of the final PEAT library.
Generation of ditag by MmeI digestion
Step 29 and 30
RCA product and enzyme should be mixed at indicated ratio to ensure efficient digestion. More recovery of desired band (~94bp) reduces the bias of the final PEAT library.
Purification of MmeI digested small fragment
Step 31
Use large vertical gel to separate the desired band (94bp); this will increase the specificity of the final PEAT library. A small fraction of undigested RCA product will be introduced in the 94bp band during gel cut, thus large gel enables more precious separation.
Total RNA is not of high quality:
Use RNase-free reagent and RNase-free water to isolate total RNA. All microtubes should be autoclaved and UV treated. Use RNaseZap RNase Decontamination Solution (Ambion) to wipe benchtop and all pipettes.
RCA is not efficient:
Multiple reasons may cause this problem. One possibility is less circular DNA is generated during circularization step. Always do quality control step 2 to make sure cDNA can be specifically amplified by primer pair Mme_F and Mme_R. Another reason is Phi29 DNA polymerase might be inactivated by UV treatment if the temperature gets too high during treatment. To solve this, make sure the PCR cap which contains enzyme meets cooled water in the well of the PCR cooler during the treatment. After adding water to the well of the PCR cooler, wait for at least 1 min to let the water to cool down to ~0 °C.
Sanger sequencing revealed high percentage of clones without built-in linkers:
The built-in linkers are used to distinguish if the 20 nt tag is derived from the TSS side or from the 3’ downstream region. Unsuccessful separation of the 94 bp band from background is the main reason. To resolve this problem, always remember to use large acrylamide gel to better separate the intended band from background. In addition, cut the ~94 bp band as tight as possible. All these efforts will reduce the background to less than 5%.
Deep sequencing results revealed that 90% of the paired reads have built-in linkers. Of those paired reads, 76% were mapped to a unique location within Release 5 of the fly genome. An additional 10% of pairs mapped to multiple genomic locations. The median distance between the 5’ and 3’ reads at the transcript level was 279 nt. 81.5% of genes currently annotated by FlyBase (v5.14) were represented by at least one read-pair.
- Collart, M.A. & Oliviero, S. Preparation of yeast RNA. Current protocols in molecular biology / edited by Frederick M. Ausubel et al., Chapter 13, Unit13 12 (2001).
- Zhang, K. et al. Sequencing genomes from single cells by polymerase cloning. Nature biotechnology 24, 680-686 (2006).
- Shendure, J. et al. Accurate multiplex polony sequencing of an evolved bacterial genome. Science 309, 1728-1732 (2005).
We are grateful to David MacAlpine and Sara Powell (Duke University Medical Center) for their help in collecting fly embryos. We thank Bin Xie (Virginia Commonwealth University) for his effort in performing the Illumina paired-end sequencing. We are indebted to Dr. Yongde Bao (University of Virginia) for his effort in optimizing the paired-end sequencing procedure.
Associated Publications
A paired-end sequencing strategy to map the complex landscape of transcription initiation
by Ting Ni, David L Corcoran, Elizabeth A Rach, +5
Nature Methods (22 May, 2012)