DNA TEMPLATE GENERATION
Experimental design (Figure 2)
The DNA template contains a T7 promoter (TAATACGACTCACTATAG), a Ribosome Binding Site (RBS - AGGAGG), a sequence of interest (random ORF, etc.), two TGA stop codons, and an EcoRV restriction site located near the stop codons. EcoRI and BamHI restriction sites are present at the 5’ and 3’ ends to allow insertion of the template into a plasmid, if needed.
The DNA template is generated by PCR using 5 oligonucleotides: Fwd_short, Fragment1_fwd, Fragment_rev, TGA_EcoRV_rev_short, and a forward oligonucleotide containing the sequence of interest (e.g. GGA-GGA-AAA-AAT-ATG-(NNN)15-GCG-ATC-TCG-GTG-TGA for the NNN-15 library)
TBE Gel preparation and polymerization TIMING 1.5 hours
1/ Prepare a 30 mL 9 % or 12 % TBE-Acrylamide gel (depending on the template length) for 5 x 15-well gels.
! CAUTION Gels must be prepared under the fume-hood because APS, TEMED and acrylamide are very toxic and irritant.
Mix the following in a 50 mL Falcon tube:
9 mL or 6.75 mL of a 40 % 19:1 acrylamide:bisacrylamide solution (for a 12 % or 9 % gel, respectively)
3 mL 10X TBE
18 mL or 20.25 mL distilled H2O (for a 12 % or 9 % gel, respectively)
120 μL APS
(Mix)
30 μL TEMED
2/ CRITICAL STEP Mix gently by inverting 5-6 times and immediately pour 6 mL per 15-well gel
3/ Wait 1 hour for polymerization
Gels can be wrapped in damp paper and stored at 4 °C. They should be used within 2 weeks.
PCR reaction TIMING 1 hour
4/ Prepare the following PCR Mix:
5 μL 5X HF Phusion buffer
0.5 μL dNTPs (10 mM stock)
0.5 μL Fwd_short (10 μM stock)
0.5 μL PURE_frag1_fwd (1 μM stock)
0.5 μL Template oligo (1 μM stock)
0.5 μL Frag2_rev_TGA_EcoRV (1 μM stock)
0.5 μL TGA_EcoRV_short (10 μM stock)
0.5 μL Phusion polymerase
16.5 μL H2O up to 25 μL
5/ Run the following PCR Program
Initial Denaturation 98 °C 30”
Denaturation 98 °C 10”
Annealing 62 °C 5”
Elongation 72 °C 10” Go 6-24x to step 2 : CRITICAL STEP, depends on the template
Hold 4 °C ∞
If the PCR is successful, produce large amounts of DNA for in vitro transcription by running PCR reactions with a combined volume of 1600 μL (50 μL reactions in 4 strips of 8 tubes). Pool 8 tubes/column for PCR purification. Ideally, a minimum concentration of 125 ng/μL should be used for in vitro transcription. Use a SpeedVac if necessary to concentrate DNA.
PAUSE POINT Samples can be frozen at -20 °C until the next step.
Analysis of PCR products TIMING 1.5 hour
6/ Sample preparation: 1 μL PCR product + 4 μL H2O + 1 μL 6X Loading Dye
7/ Loading: 0.5 μL of molecular weight ladder and 5 μL of each sample
8/ Gel electrophoresis conditions: 1 hour at 200 V in 1X TBE solution
9/ Staining: Soak the gel in 1X SybrGold for 15 minutes
CRITICAL STEP Do not use the same SybrGold solution more than 4 times. Prepare a fresh solution if the gel is not sufficiently stained.
! CAUTION Be sure to wear gloves and a lab coat
10/ Imaging: Use Image Lab software to visualize the stained gel
! CAUTION Acrylamide gels with or without SybrGold must be discarded in dedicated trash buckets for toxic waste
If the desired PCR product is obtained, estimate the amount of DNA to purify on QiaQuick column(s)
PAUSE POINT Samples can be frozen at -20 °C until the next step.
PCR purification and PCR product concentration assessment TIMING 30 minutes
11/ See the instructions manual provided by the manufacturer of the QiaQuick PCR Purification Kit
from Qiagen®
- 10 μg max. DNA should be loaded onto a single column for 80 % final recovery
- 700 μL for capacity of column reservoir (load/spin multiple times if volume >700 μL)
CRITICAL STEP Wash at least two times with PE-buffer and dry the column with an additional centrifugation
- Elute in 30 μL H2O
12/ Use the Nanodrop to determine the concentration of PCR product. Pure DNA should have an A260/280 ratio of 1.8 and an A260/230 ratio between 1.8 and 2.2
PAUSE POINT Samples can be frozen at -20 °C until the next step.
IN VITRO TRANSCRIPTION
Experimental design (Figure 3)
The mRNA is transcribed in vitro from the DNA template yielding an mRNA fragment containing an RBS, a region of interest, spacers, two stop codons (UGA) and the EcoRV restriction site.
TBU Gel preparation and polymerization TIMING 2.5 hours
13/ Prepare a 30 mL 9 % TBU-Acrylamide gel solution for 5 x 15-well gels
! CAUTION Gels must be prepared under the fume-hood because APS, TEMED and acrylamide are very toxic and irritant
In a 50 mL Falcon tube, mix:
14.4 g Urea
6.75 mL Acrylamide 19:1 40 %
6 mL 10X TBE
Distilled H2O up to 30 mL
CRITICAL STEP Mix gently on rotator to completely dissolve the urea and add:
60 μL APS
Mix by inverting
33 μL TEMED
Mix by inverting
Pour immediately 6 mL per 15-well gel prior to polymerization
14/ Wait 1 hour for polymerization
Gels can be stored at 4 °C in damp tissue and an airtight bag. They should be used within 2 weeks.
In vitro transcription reaction TIMING 4 hours
In vitro transcription with a new template has to be tested in a small volume reaction (20 μL) according to the manufacturer’s protocol, before being scaled up to 60 μL if needed.
15/ Mix:
10 μL RiboMAX™ Express T7 2X Buffer (contains NTPs)
2 μL Enzyme Mix T7Express
X μL DNA template (1 μg equivalent to a final concentration of 50 ng/μL)
X μL H2O up to 20 μL
16/ Keep a 1 μL aliquot from the reaction at t=0 and DNA template as controls
17/ Incubate at 37 °C in a dry incubator or thermomixer for 3 hours
OBSERVATIONS Frozen RiboMAXTM Express T7 2X Buffer will contain a precipitate that can be dissolved by warming the buffer at 37 °C and mixing well
A white precipitate indicating production of pyro-phosphate can be observed.
18/ Spin down the reaction to pellet the pyro-phosphate 30 minutes at full speed at 4 °C and transfer the supernatant containing the mRNA into a new vial.
CRITICAL STEP Do NOT freeze transcription reactions. After the transcription reaction is complete, proceed directly to the DNase step and removal of unincorporated NTPs
DNase I treatment of in vitro transcribed mRNA TIMING 30 minutes
CRITICAL STEP All the following steps should be carried out under RNase-free conditions. Use RNase-free tubes, pipettes, filter tips and water. Wear gloves at all times.
19/ Add 1 μL of RQ1 RNase-free DNase I (eq. 1 unit per microgram of template DNA)
20/ Incubate 15 minutes at 37 °C in thermomixer or dry incubator
Purification and electrophoretic analysis of in vitro transcribed mRNA TIMING 30 minutes
Purification of mRNA >17 nt according to the manufacturer’s protocol of RNA Clean & ConcentratorTM-5 from Zymo Research.
Perform all steps at room temperature and centrifugation at 10,000 to 16,000 x g for 30 seconds, unless specified.
21/ Before processing, adjust the sample volumes to at least 50 μL: 20 μL reaction volume + 30 μL H2O
22/ Add 2 volumes of RNA Binding Buffer to each sample and mix (e.g. mix 100 μL buffer with 50 μL sample)
23/ Add an equal volume of ethanol (95-100 %) and mix (e.g. add 150 μL ethanol)
+IMPROVEMENTS Alternatively, if the sample consists of RNA species 17-200 nt, use 1.5 volumes of ethanol
24/ Transfer the sample to the column in a collection tube and centrifuge.
25/ Discard the flow-through
26/ Add 400 μL RNA Prep Buffer to the column and centrifuge. Discard the flow-through
27/ Add 700 μL RNA Wash Buffer to the column and centrifuge. Discard the flow-through
28/ Add 400 μL RNA Wash Buffer to the column and centrifuge for 2 minutes to ensure complete removal of the wash buffer. Carefully transfer the column into a RNase-free tube (not provided)
29/ Add 15 μL RNase-free water directly to the column matrix and centrifuge.
+IMPROVEMENTS Perform an additional elution step (15 μL) in a new tube to recover more RNA
OBSERVATIONS Here, even if the maximal recovery is 10 μg total RNA we can often obtain between 10 and 70 μg mRNA in the end.
PAUSE POINT Samples can be frozen at -20 °C or -80 °C until the next step.
30/ Use the Nanodrop to determine the mRNA concentration (1:10 dilution in H2O). Pure RNA has an A260/280 ratio of 2 and an A260/230 ratio between 1.8 and 2.2. Use this dilution for subsequent analysis on a TBU gel.
31/ Pre-run the 9 % TBU-Acrylamide gel for about 20 minutes at 200V in 1X TBE. This will result in a nicer looking gel.
32/ Sample preparation: add the volume corresponding to 25-50 ng of mRNA to 15 μL RNA Loading Buffer
33/ Heat samples and the RNA molecular weight ladder for 5 minutes at 95 °C
34/ Load 15 μL of RNA molecular weight ladder and the entire amount of each sample
CRITICAL STEP Remember to flush out the wells with a pipette or a syringe just prior to loading your samples in order to remove urea.
34/ Gel electrophoresis conditions: 1 hour at 200 V
35/ Staining: Soak the gel for 15 minutes in SybrGold
36/ Imaging: Use Image Lab software to visualize the stained gel
! CAUTION Clean electrophoresis plates with soap and rinse with distilled water. Never use ethanol.
Acrylamide gels with or without SybrGold must be discarded in dedicated trash buckets for toxic waste.
If mRNA is correctly transcribed, scale up the reaction to 60 μL.
? TROUBLESHOOTING If the product does not look good on the TBU gel, load less mRNA on the gel or try to vary the DNA template concentration in the reaction first.
PAUSE POINT Samples can be frozen at -20 °C or -80 °C until the next step.
5’-BIOTINYLATION OF mRNA
Experimental design (Figure 4)
In order to biotinylate the 5’-end of mRNA, in vitro transcripts are treated with 3’-biotin GTP (mRNA:3’-Biotin-GTP ratio of 1:100 ) and Vaccina Capping Enzyme.
Capping of mRNA at 5’-end mRNA with 3’-Biotin-GTP and Vaccinia Capping enzyme TIMING 45 minutes
The optimal mRNA:3’-Biotin-GTP ratio for biotinylation is 1:100. Usually, 200 pmol of mRNA can be biotinylated with 20,000 pmol of 3’-Biotin-GTP in a 20 μL reaction volume. This volume can be scaled-up, depending on the number of iTP-seq reactions (5 pmol of mRNA/condition).
37/ In a tube, mix:
X μL mRNA template (200 pmol)
X μL H2O up to 12 μL
38/ Incubate 5 minutes at 65 °C in thermomixer
39/ Add in the following order :
2 μL 10X Buffer
4 μL 3’-Biotin-GTP 5mM (20 000 pmol)
2 μL Vaccinia Capping Enzyme (20 units)
40/ Incubate 30 minutes at 37 °C in thermomixer
Purification of biotinylated mRNA TIMING 30 minutes
Purification of mRNA >17 nt according to the manufacturer’s protocol of RNA Clean & Concentrator™-5 from Zymo Research.
Perform all steps at room temperature and centrifugation at 10,000 to 16,000 g x g for 30 seconds, unless specified.
41/ Before processing, adjust the sample volumes to at least 50 μL: 20 μL reaction volume + 30 μL H2O
42/ Add 2 volumes RNA Binding Buffer to each sample and mix (e.g. mix 100 μL buffer with 50 μL sample)
43/ Add an equal volume of ethanol (95-100 %) and mix (e.g. add 150 μL ethanol)
+IMPROVEMENTS Alternatively, if the sample consists of RNA species 17-200 nt, use 1.5 volumes of ethanol
44/ Transfer the sample to the column in a collection tube and centrifuge.
45/ Discard the flow-through
46/ Add 400 μL RNA Prep Buffer to the column and centrifuge. Discard the flow-through
47/ Add 700 μL RNA Wash Buffer to the column and centrifuge. Discard the flow-through
48/ Add 400 μL RNA Wash Buffer to the column and centrifuge for 2 minutes to ensure complete removal of the wash buffer. Carefully transfer the column into an RNase-free tube (not provided)
49/ Add 15 μL RNase-free water directly to the column matrix and centrifuge.
+IMPROVEMENTS Perform an additional elution step (15 μL) in a new tube to recover more RNA
OBSERVATIONS Here, even if the maximal recovery is 10 μg total RNA we can often obtain between 10 and 70 μg of mRNA in the end.
PAUSE POINT Samples can be frozen at -20 °C or -80 °C until the next step.
50/ Use the Nanodrop to determine the mRNA concentration (1:10 dilution in H2O). Pure RNA has an A260/280 ratio of 2 and an A260/230 ratio between 1.8 and 2.2.
Check incorporation and efficiency of biotinylation with Dot Blot TIMING 1 day
51/ Switch on the oven at 80 °C
52/ Cut out a piece of Hybond+ membrane, enough for 5-6 spots of the standard plus the number of samples to analyze. CRITICAL STEP Do not touch the membrane directly with your hands. Instead, use tweezers to handle it.
53/ Pre-wet the membrane in 6X SSC buffer (can be reused) for 10 minutes
54/ In the meantime, prepare the standard and the samples (dilute in 6X SSC buffer). 1 μL of each sample and standard will be placed on the membrane, meaning that dilutions with the required quantity of nucleic acids in 1 μL need to prepared.
CRITICAL STEP Dilutions are made in 6X SSC buffer
- Standard: “Biotin-Linker” biotinylated oligonucleotide (100 pmol/μL stock)
Serial dilutions:
S1: 0.5 μL Biotin linker + 4.5 μL 6X SSC Final concentration at 10 pmol/μL
S2: 2.5 μL from S1 + 2.5 μL 6X SSC Final concentration at 5 pmol/μL
S3: 2.5 μL from S2 + 2.5 μL 6X SSC Final concentration at 2.5 pmol/μL
S4: 2.5 μL from S3 + 2.5 μL 6X SSC Final concentration at 1.25 pmol/μL
S5: 2.5 μL from S4 + 2.5 μL 6X SSC Final concentration at 0.625 pmol/μL
- Sample:
Serial dilutions:
R1: x μL Biotinylated mRNA + y μL 6X SSC Final concentration at 5 pmol/μL
R2: 2.5 μL from R1 + 2.5 μL 6X SSC Final concentration at 2.5 pmol/μL
R3: 2.5 μL from R2 + 2.5 μL 6X SSC Final concentration at 1.25 pmol/μL
R4: 2.5 μL from R3 + 2.5 μL 6X SSC Final concentration at 0.625 pmol/μL
55/ Draw a grid on a piece of Whatman paper corresponding to the size of the membrane, with enough room to spot the standards and the samples
56/ Place the pre-wetted membrane onto the grid
57/ Mark outer grid ---- positions and X in one corner on the membrane with a soft pencil
58/ Pipette the standards and samples onto the membrane according to the grid
CRITICAL STEP Do not touch the membrane with the pipette tip and keep the remaining samples for analysis on a TBU gel.
59/ Cover membrane with a second piece of Whatman paper, wrap in aluminum foil and bake at 80 °C for 2 hours. If you are in a rush, this can be reduced to 1 hour
60/ Blocking: Place membrane into a 50 mL Falcon tube and block membrane with 50 mL TBS-T + 2.5 g milk powder. Incubate for 1 hour on tube rotator at room temperature
CRITICAL STEP Take care that the whole membrane is covered with the solution.
PAUSE POINT Samples can be kept in milk O/N.
61/ Discard TBS-T-milk solution
62/ Hybridization: Incubate the membrane with 12.5 mL of a 1:2500 dilution of Streptavidin-alkaline Phosphatase in TBS-T for 1 hour on rotating wheel
63/ Discard the Streptavidin-alkaline Phosphatase solution
64/ Wash membrane 3 x 10 minutes with TBS-T buffer
65/ For detection, mix:
10 mL Alkaline Phosphatase buffer
66 μL NBT (Mix by inverting)
33 μL BCIP (Mix by inverting)
CRITICAL STEP Always mix components in this order, otherwise detection will not work
66/ Check the labeling within 10-20 minutes
67/ Discard detection solution and wash membrane once or twice with TBS-T to stop the reaction
68/ Take a picture of the spots soon after detection: Biorad imager ->Protocol: Blot -> colorimetric ->No filter
Analysis of biotinylated mRNA TIMING 2 hours (optional)
69/ Pre-run the gel for approximately 20 minutes at 200V in 1X TBE
70/ Sample preparation: 25-50 ng of diluted biotinylated mRNA solution used for the Dot Blot + 15 μL RNA Loading Buffer
71/ Heat samples and RNA molecular weight ladder (15 μL) for 5 minutes at 95 °C
CRITICAL STEP Remember to flush out the wells with your pipette tip or a syringe just prior to loading your samples. The samples will run nicer on the gel.
72/ Load 15 μL of RNA molecular weight marker and the full volume of each sample
73/ Run the gel: 1 hour at 200 V
74/ Staining: Soak the gel for 15 minutes in SybrGold
CRITICAL STEP Do not use the same SybrGold solution more than 4 times. Prepare a fresh solution if the gel is not sufficiently stained.
! CAUTION Be sure to wear gloves and a lab coat
75/ Imaging: Use the Image Lab software to visualize the stained gel
76/ Cleaning: Clean electrophoresis plates with soap and rinse with distilled water
! CAUTION Acrylamide gels with or without SybrGold must be discarded in dedicated trash buckets for toxic waste.
+IMPROVEMENTS For all subsequent steps, make pre-mixes for n+1 tubes where appropriate in order to improve reproducibility and save time
IN VITRO TRANSLATION
Experimental design (Figure 5)
We use the PURExpress® system to translate biotinylated mRNAs in vitro. This reconstituted protein expression system is based on the PURE system (Ref.4: Shimizu, Y et al. 2001. Nat Biotechnol) and contains all of the components needed for in vitro transcription and translation.
In vitro translation reaction TIMING 1 hour (depending on the number of samples and without the time required to prepare aliquots)
CRITICAL STEP Thaw kit solutions on ice just before use and keep on ice at all times. Make 10 μL aliquots of each kit component, each of which will subsequently be used for a single reaction. Freeze unused aliquots in liquid N2 and store at -80 °C. It is advisable to plan ahead and think about how many aliquots you will need when opening a new kit.
OBSERVATIONS There are different types of PURExpress kits. Use the one most suitable for your experiments and refer to its specific protocol. Note that the choice of release factors to use will depend on the nature of the stop codons present in your library. RF1 recognizes UAA and UAG. RF2 recognizes UAA and UGA. RF3 helps RF1 and RF2 to dissociate from the ribosome.
CRITICAL STEP Never spin PURExpress components in the table top centrifuge. Instead use a refrigerated minicentrifuge.
CRITICAL STEP Antibiotics, small molecule ligands or protein factors may be added to the mix according to your needs. Small molecule ligands are typically first dried inside the tube using a SpeedVac.
Reactions can be performed in a 5 μL reaction volume with 5 pmol of biotinylated mRNA.
Control reactions (i.e. lacking an antibiotic, small molecule or protein factor) are typically prepared at this step.
A 5 μL PURExpress reaction contains 12 pmol of ribosomes. Use 5 pmol of mRNA to ensure an excess of ribosomes over mRNA.
77/ Take the number of aliquots of each kit component corresponding to the desired number of samples out of the freezer
78/ Pool all of the aliquots for each component
79/ Make a master mix
80/ Assemble the reaction on ice in a new tube in the specific order indicated in the protocol for a final volume of 5 μL for each reaction
81/ Incubate reactions for 30’ at 37 °C in the incubator
82/ Immediately after incubation, place the in vitro translation reactions on ice
CRITICAL STEP It is crucial to go on to the next step immediately and not to pause the protocol at this stage!
RNASE R DIGESTION
Experimental design (Figure 6)
Truncated RNase R degrades mRNA from the 3’-end. When RNase R encounters the leading ribosome on an mRNA, it drops off the mRNA and a ribosome-protected inverse toeprint is generated.
CRITICAL STEP Adding ice-cold buffer to increase the Mg2+ concentration to 50 mM stabilizes ribosomes on the mRNA. Think to thaw this buffer on ice 1 hour before you use it and make sure it is cold upon addition.
RNase R reaction TIMING 45 minutes
83/ Mix:
5 μL In vitro translation reaction
5 μL RNase R Ice-cold buffer
1 μL RNase R (Stock concentration at 3 mg/mL)
It is possible, depending on the reaction number, to do a pre-mix of Ice-cold buffer and RNAse R and distribute 6 μL per tube
84/ Mix by pipetting and avoid foaming the sample
85/ Incubate 30’ at 37 °C in thermocycler (incubate mode) or in bacterial incubator
CRITICAL STEP Resuspend digested mRNA in 140 μL of 1X BWT buffer used in the following step. The NaCl contained in this buffer will (i) ensure that the ribosomes dissociate, and (ii) inhibit RNase R. There is no need for further purification.
PAUSE POINT Samples can be kept at -20 °C or -80 °C until the next step.
mRNA PURIFICATION ON DYNABEADS M-280
Experimental design (Figure 7)
Streptavidin-coated Dynabeads are used to specifically fish out the biotinylated mRNA.
CRITICAL STEP Always use low binding tubes and tips to prevent Dynabeads from sticking to the tube or pipette tip.
Washing of Dynabeads and Binding TIMING e.g. 1h45 minutes for 13 samples
OBSERVATIONS The maximal binding capacity of M-280 Dynabeads is around 23 pmol per 10 μL.
1 M NaCl is necessary for minimize non-specific binding to the beads.
1 wash step consists of :
• Resuspending the beads
• Placing the tube on a magnetic rack
• Waiting for 1 minute
• Removing the supernatant
CRITICAL STEP All steps are performed at room temperature and incubation of Dynabeads is always performed on a tube rotator (Rotoflex) to stop the beads from settling at the bottom of the tube.
CRITICAL STEP Wash tubes one by one to avoid drying the samples and to prevent cross-contamination.
86/ Vortex Dynabeads for at least 30 seconds in order to resuspend them
87/ Dispense a suitable volume of Dynabeads into a fresh tube (use 5 μL bead suspension per in vitro translation reaction with 5 pmol of mRNA)
88/ Add an equal volume (or at least 500 μL) of 1X BWT buffer and mix
89/ Place the tube on the magnetic rack and wait for at least 1 minute while beads collect on the side of the tube
90/ Leaving the tube on the magnetic rack, carefully remove the supernatant by pipetting on the side of the tube opposite to where the beads are collected
91/ Remove the tube from the magnetic rack and resuspend the beads in 500 μL of 1X BWT buffer
92/ Perform 2 additional washes with 500 μL 1X BWT buffer
93/ Resuspend beads in 1X BWT buffer in order to have 50 μL/reaction (ex. For 2 reactions resuspend in 100 μL)
94/ Combine biotinylated mRNA sample (approximate volume of 150 μL) with 50 μL of washed Dynabeads
95/ Binding: Resuspend thoroughly and incubate at room temperature on tube rotator (Rotoflex) for 15 minutes
96/ Wash beads twice with 500 μL 1X BWT buffer
97/ Wash beads twice with 500 μL H2O
98/ Remove all liquid from the tube after the last washing step
99/ Perform a quick spin in a minifuge and put on magnetic rack for 1 minute to remove all of the residual liquid
100/ Resuspend the beads in 4 μL H2O. Together with the volume of the beads this will give a combined volume of 4.5 μL
PAUSE POINT Samples can be stored at 4 °C until the next step. The shorter they stay in the fridge, the better.
LINKER LIGATION
Experimental design (Figure 8)
The linker is adenylated at its 5’-end and carries ddC at its 3’-end. The adenylation enables ligation of the ssDNA to the mRNA, whereas the ddC stops the linker from being circularized.
Adding the linker to the 3’-end of the purified mRNA introduces a known 3’-sequence needed for subsequent reverse transcription.
CRITICAL STEP For the linker ligation step, make a pre-mix, ensuring proper mixing of all component in the reaction tube, especially 50% PEG 8000, buffers and DTT.
Ligation TIMING 2h30
OBSERVATIONS Make sure that you use the correct linker. For example, if there is an EcoRV restriction site in your construct, use the linker containing the ApoI restriction site (usually, efficiency not higher than 80%) and vice versa. Use of a master mix is advisable.
101/Mix for 1 reaction:
1 μL Linker ApoI or EcoRV (10μM stock)
1 μL 10X T4 RNA ligase 2, truncated Buffer
3 μL 50% PEG 8000 (Quite viscous, pay attention when pipetting)
0.5 μL T4 RNA ligase 2, truncated (200 000 U/mL stock)
Add to 4.5 μL Dynabeads covered with mRNA
CRITICAL STEP To improve reliability do a master mix for n+1 samples in order to avoid pipetting errors related to viscous reagents. Distribute 5.5 μL of pre-mix per tube
102/ Mix the ligation reaction thoroughly with a 10 μL pipette
103/ Incubate the reaction on the tube rotator (Rotoflex) for 2 hours at room temperature
Washing TIMING e.g. 1h for 13 samples
OBSERVATIONS Switch on the thermomixers to 55°C and 65°C for the reverse transcription step
104/ Wash the beads twice with 500 μL H2O
105/ Remove all liquid from the tube after the last washing step
106/ Spin tubes in a minifuge and put on a magnetic rack for 1 minute to remove all residual liquid
107/ Resuspend the beads in 11.5 μL H2O
PAUSE POINT Samples can be stored at 4°C until the next step. The shorter the better.
REVERSE TRANSCRIPTION
Experimental design (Figure 9)
The reverse transcription step generates the DNA complementary to the mRNA (cDNA). It will be possible to perform a PCR on this cDNA to amplify the ribosome-protected sequences.
Reverse Transcription TIMING 1 hour
CRITICAL STEP For reverse transcription, make a pre-mix and use 2 μL of it in each tube.
108/ For 1 reaction, mix:
1 μL dNTPs (10mM stock)
1 μL oligo miRNA linker rev (2 μM stock)
Add to 12 μL Dynabeads coated with mRNA-linker
109/ Thoroughly mix and incubate for 5’ at 65 °C at 500 rpm in the thermomixer to anneal the primer to the complementary sequence
110/ Place tube(s) on ice for a couple of seconds
111/ Add 6 μL of the following mix to each tube:
4 μL 5X First Strand synthesis buffer (5X FS)
1 μL DTT 0.1 M stock
1 μL SuperScript™ III Reverse Transcriptase
Thoroughly mix and incubate for 30 ’ at 55°C at 500 rpm in the Thermomixer.
OBSERVATIONS To prevent settling of the Dynabeads, you can mix by pipetting at t= 15 min.
PAUSE POINT Samples can be stored at 4°C until the next step. The shorter the better.
FILL-UP, RESTRICTION ENZYME TREATMENT AND PCR TO AMPLIFY cDNA
Experimental design (Figure 10)
cDNA corresponding to inverse toeprints are amplified in 3 steps:
- “Fill-up” reaction with the forward primer in order to synthetize the sense strand
- +/- Restriction Enzyme treatment:
• When ribosomes stall before reaching the stop codon, the restriction site is digested by RNase R and the linker is still present after digestion. Amplification is possible by PCR and the product will be shorter than the full-length mRNA.
• When ribosomes are stalled on the stop codon due to the absence of release factor 2 (RF-2) in the translation reaction, the restriction site is protected from digestion by RNase R. The restriction enzyme can cut the DNA and the linker will be released. Consequently, cDNA will not be amplified.
• If no restriction enzyme is used, cDNAs corresponding to full-length mRNAs will also be amplified.
- PCR: cDNA that has not undergone restriction digestion is amplified.
To determine the correct number of PCR cycles for amplification, split the reaction into 4-5 tubes and test different numbers of PCR cycles (e.g. 8, 10, 12, 14). To avoid using several thermocyclers, stay next to the machine and take out the samples after the desired number of cycles.
Fill-up reaction TIMING 30 minutes
112/ Prepare a mix corresponding to the number of samples+1
For 1 sample:
10 μL 5X HF Phusion buffer
1 μL dNTPs (10 mM stock)
1 μL Oligo cDNA Forward (10 μM stock)
1 μL Phusion polymerase
35 μL H2O up to 48 μL
Add 48 μL of the mix to 1μL of reverse transcription product (dynabeads mix)
113/ Run the “Fill-Up” program
Denaturation 98°C 10”
Annealing 42°C 10”
Elongation 72°C 30”
Restriction enzyme treatment TIMING 1 hour
114/ Split into 2 tubes of 24.5 μL
115/ Add 0.5 μL of restriction enzyme to the PCR mix in the first tube and 0.5 μL of water (control) to the PCR mix in the second tube. If you added ApoI in the linker, use EcoRV-HF in this step and vice versa.
116/ Mix thoroughly by pipetting
117/ Incubate for 1h at 37 °C in the incubator
Amplification by PCR TIMING 1 hour
118/ Add 0.5 μL of the oligo miRNA linker Rev (10 μM stock). If you wish to test different numbers of cycles, you can split your PCR tubes in order to have 12.5 μL (minimum) for each PCR reaction
119/ Run the PCR program:
Initial denaturation 98°C 30”
Denaturation 98°C 10”
Annealing 42°C 10”
Elongation 72°C 10” go to step 2, x times (ex: n=12-15-18)
Hold 12°C ∞
Analysis of PCR products TIMING 1.5 hour
120/ Sample preparation: dilute 3-10 μL of PCR product in water to give a volume of 10 μL and add 2 μL of 6X Loading Dye
121/ Load 0.5 μL of Molecular weight ladder and 12 μL of each sample onto a TBE gel
122/ Gel electrophoresis conditions: 1 hour at 200 V in 1X TBE running buffer
123/ Staining: soak the gel for 15 minutes in SybrGold
CRITICAL STEP This step is important to choose the minimum number of PCR cycles required to see your product, but not the higher molecular weight contaminants / byproducts.
CRITICAL STEP Do not use the same SybrGold solution more than 4 times. Prepare a fresh solution if the gel is not sufficiently stained.
! CAUTION Be sure to wear gloves and a lab coat if you do not want to stain your own DNA
124/ Imaging: Use Image Lab software to image your gel
! CAUTION Acrylamide gels with or without SybrGold must be discarded in dedicated trash buckets for toxic waste.
PURIFICATION OF cDNA TO USE AS A TEMPLATE FOR NGS LIBRARY PREPARATION
Use the amplified cDNA from the previous step as a PCR template to add the NGS adapter for sequencing.
PCR template preparation TIMING 2 hours
125/ Once the optimal number of PCR cycles is established, run 5 x 25 μL PCR reactions (in combination with the restriction enzyme treatment) per sample as described in steps 115-122
Preparation of medium-sized 9% acrylamide gels and gel electrophoresis TIMING 20 hours
126/ Clean the glass plates thoroughly (H2O-EtOH-H2O) and dry them with Kim-wipes.
Put the blue silicone rubber joint around the larger of the two plates and place the 1.5mm spacers on the right and left sides.
Place the other plate on the first one and put the clips on (n=6/ 2 per side)
Prepare a 60 mL 9% TBE gel in two 50 mL Falcon tubes
! CAUTION Gel preparation must be performed under the fume-hood because APS, TEMED and acrylamide are very toxic and irritant
For a 9% Acrylamide gel, add the following In each 50 mL Falcon tube:
6.75 mL Acrylamide:bis-acrylamide 19:1 40%
3 mL 10X TBE
20.25 mL distilled H2O
120 μL APS
Mix by inverting
30 μL TEMED
Cast the gel (60 mL/gel), insert a 20-well comb and let it polymerize
127/ Mix 25 μL of each sample + 5 μL of 6X Loading dye, and load on the gel. Put side by side gels with and without antibiotics or ligands to further identify the band of interest
OBSERVATIONS Leave empty wells between each type of sample and between the samples and ladder. This will help minimize cross-contamination.
128/ After checking that the gel tank is level with the ground, do a pre-run to test the system and run the PCR products on medium-sized 9% acrylamide gels (Volume 60 mL) at 175 V for 18 hours. You can run two different samples side by side on a single gel separated by 2 μL of Low molecular weight DNA Ladder. Fill empty wells with 5 μL of 6X Loading dye
Recovering the bands of interest TIMING 1 hour (1 gel)
129/ Separate the gel plates and cut 1 corner of the gel to help orient it
! CAUTION Change the SybrGold in the large gel tank (after rinsing it with distilled water and EtOH after each use to avoid contamination): 2 aliquots of SybrGold (eq.10 μL) in 1X TBE
130/ Stain the gel with SYBR Gold
CRITICAL STEP To avoid cross-contamination, put Saran wrap on the UV plate and prepare in advance 1 fresh scalpel, 1 syringe and 1 x 15 mL Falcon tube per sample.
131/ Take a picture with the imager in order to see which part of the gel you will cut
132/ Place the Plexiglas plate on the drawer of the imager. Switch off the light inside the gel room and switch on the UV transilluminator.
133/ Cut out the band(s) of interest with a clean scalpel. For example if you are using an NNN or NNS library, cut the gel to include the region between the top edge of the start codon band and the stop codon band. Including too much of the start codon band will yield too many reads for initiation complexes at the expense of reads for elongation complexes.
134/ Remove the plunger from a 5 mL syringe, put the gel pieces into the syringe, and put the plunger back in
135/ Take a picture of the gel after cutting the bands out to check that you took all of DNA of interest
136/ Crush the gel pieces by pushing them through the opening of the syringe and store them in a 15 mL Falcon tube
137/ Add 10 mL of gel extraction buffer in Falcon tubes containing gels debris. Rinse the syringe, plunger and scalpel with this buffer to avoid losing part of your samples
CRITICAL STEP Make sure that all of the gel debris is at the bottom of your tubes.
Extract the DNA by diffusion TIMING 24-48 hours
138/ Leave samples on tube rotator at room temperature for at least 24 hrs, or over the weekend
139/ Pre-wet a 0.22 μm Vivaspin filter with 6 mL of gel extraction buffer and centrifuge 1 min. at 4,000 x g at 4°C (ThermoFisher Multifuge X3R- Rotor Fiberlite F15 with small adapters)
140/ Filter the supernatant through the 0.22 μm Vivaspin column (6 mL volume). Do 2 successive centrifugations for 5 minutes at 4,000 x g. After each centrifugation, collect the flow-through in a fresh 15 mL Falcon tube. Recover the supernatant and liquid around the acrylamide debris with a 1 mL pipette
! CAUTION Here, tubes and samples are contaminated with SybrGold. Use good laboratory practices for handling and disposal of samples.
PAUSE POINT Samples can be stored at -20 °C until the next step. The volume should be approximately 10 mL per sample.
Isopropanol precipitation of inverse-toeprinting samples TIMING 30 minutes
141/ Split the ~10 mL of each sample into 6 x 2 mL low-binding Eppendorf tubes (~1.5 mL per tube)
142/ Concentrate DNA using a SpeedVac (H2O mode-50 °C) to reach a volume of ~200 μL in each tube (expected time of evaporation, around 3 hours)
143/ Pool all fractions together (~1.1 mL) and add GlycoBlue reagent
Initial concentration: 15000 μg/mL. Final concentration: 50 μg/mL. Final volume: 1100 μL -> 3.7 μL GlycoBlue reagent/sample
144/ Add 1 volume of isopropanol (here, 1 mL)
145/ Vortex and leave on ice for 15 minutes
146/ Store at -80°C O/N
PAUSE POINT Samples can be stored at -80°C until the next step.
Recovery of precipitated DNA TIMING 2 hours
147/ Spin DNA at 20,000 x g for 30 minutes at 4°C in ThermoScientific centrifuge with rotor for 50 mL Falcon tubes and small adaptors
148/ Dry the pellet (approx. 1h on bench) after carefully discarding the supernatant and dry tube walls with clean Kim-wipes
149/ Resuspend the pellet of cDNA in 20 μL of H2O
PAUSE POINT Samples can be stored at -20°C until the next step.
ADDITION OF NGS ADAPTERS AND LIBRARY PREPARATION
Experimental design (Figure 11)
The goal of this step is to add TruSeq DNA adapters by PCR. The forward primer is universal while the reverse primer contains a specific barcode. DNA from each experimental condition will be amplified with a specific barcoded reverse oligonucleotide, enabling multiplexing during next-generation sequencing (Illumina).
All samples will be mixed in the desired molar ratios prior to next-generation sequencing.
PCR optimization TIMING 1.5 hours
OBSERVATIONS PCR amplification of cDNA should be optimized by running different numbers of cycles (e.g. 12, 14, 16, 18). Previous experiments with NNN or NNS libraries showed that running fewer PCR cycles yields more specific PCR products with the expected size distribution.
150/ Carefully choose the barcoded reverse primers with different index numbers / frameshifts. 1 sample with 1 index corresponding to 1 specific barcode.
151/ PCR mix for 1 sample:
10 μL 5X HF Phusion buffer
1 μL dNTPs (10 mM stock)
1 μL Oligo Forward NGS universal (1 μM stock)
1 μL NGS_barcode_Reverse (1 μM stock)
1 μL NGS_universal_Forward_short (10 μM stock)
1 μL NGS_universal_Reverse_short (10 μM stock)
0.5 μL Purified cDNA
1 μL Phusion polymerase
x μL H2O up to 50 μL
+OBSERVATIONS Make a master mix for (n+1) samples with all of the components listed in step 151, with the exception of the sample-specific NGS_barcode_Reverse oligonucleotide and cDNA which can be added individually afterwards. Distribute the mix in tube containing sample-specific NGS_barcode_Reverse oligonucleotide and corresponding cDNA.
152/ Split PCR mix into 4 tubes (12.5 μL each), one for each of the number of cycles tested
153/ Run the following program in the thermocycler:
Initial denaturation 98°C 30”
Denaturation 98°C 10”
Annealing 42°C 10”
Elongation 72°C 10” Go to step 2 and repeat 11 to 17 times.
Hold 12°C ∞
PAUSE POINT Samples can be frozen at -20°C until the next step.
Purification and analysis of PCR products on TBE gel TIMING 2.5 hours
154/ Prepare a 12% TBE acrylamide gel
155/ Add 3 μL of 5X loading dye to each PCR reaction and load 10 μL on the gel, along with 0.5 μL Molecular weight ladder
156/ Gel electrophoresis conditions: 1 hour at 200 V in 1X TBE running buffer
157/ Staining: soak the gel for 15 minutes in SybrGold
CRITICAL STEP Do not use the same SybrGold solution more than 4 times. Prepare a fresh solution if the gel is not sufficiently stained.
! CAUTION Be sure to wear gloves and a lab coat if you do not want to stain your own DNA
158/ Imaging: Use Image Lab software to image your gel.
! CAUTION Acrylamide gels with or without SybrGold must be discarded in dedicated trash buckets for toxic waste.
CRITICAL STEP Choose the minimum number of cycles required to see your product, but not the higher molecular weight contaminants / byproducts.
Addition of TruSeq DNA adapters by PCR TIMING 1.5 hours
159/ Repeat the PCR for all samples using the optimized number of cycles, as described in Steps 151-153
PAUSE POINT Samples can be frozen at -20 °C until the next step.
Purification and analysis of PCR products on TBE gel TIMING 2.5 hours
160/ For PCR product purification using spin columns, please refer to the instruction manual provided by the manufacturer of the PCR Purification Kit
from Qiagen®, Macherey Nagel or Eurogentec
- Elute DNA in 30 μL H2O
161/ Measure DNA concentration on the Nanodrop.
162/ Prepare a 12% TBE-acrylamide gel. Mix a volume of PCR product corresponding to 50 ng of DNA with x μL H2O to yield a combined volume of 5 μL. Add 1 μL 6X Loading Dye and load samples on the gel, along with 0.5 μL of Molecular weight ladder
163/ Gel electrophoresis conditions: 1 hour at 200 V in 1X TBE running buffer
164/ Staining: soak the gel for 15 minutes in SybrGold
CRITICAL STEP Do not use the same SybrGold solution more than 4 times. Prepare a fresh solution if the gel is not sufficiently stained.
! CAUTION Be sure to wear gloves and a lab coat if you do not want to stain your own DNA
165/ Imaging: Use Image Lab software to image your gel
! CAUTION Acrylamide gels with or without SybrGold must be discarded in dedicated trash buckets for toxic waste.
PAUSE POINT Samples can be frozen at -20 °C until the next step.
Preparation of input mRNA library for NGS by RT-PCR
OBSERVATIONS Sequencing your input mRNA library can be a good idea in order to assess whether it has the desired complexity. Sequencing it requires you to reverse transcribe and amplify your biotinylated mRNA library.
Reverse-transcription TIMING 1 hour
166/ Dilute your biotinylated mRNA library to 5 pmol/μL
167/ Reverse-transcription reaction mix:
1 μL Biotinylated mRNA (5 pmol/μL stock)
1 μL dNTPs (10 mM stock)
4 μL 5X First Strand synthesis buffer (FS 5X)
1 μL DTT 0.1 M stock
5 μL NGS_barcode_Reverse primer (1 μM stock)
7 μL H2O (up to 19 μL)
168/ Heat reaction mix for 5 minutes at 65°C to anneal the NGS_barcode_Reverse primer to its complementary mRNA sequence
169/ Place tube on ice for a couple of seconds
170/ Add 1 μL of Superscript III enzyme
171/ Thoroughly mix by pipetting and incubate 30 minutes at 55 °C in thermomixer with shaking (500 rpm)
PAUSE POINT Samples can be frozen at -20°C until the next step.
PCR TIMING 2 hours (depending of the number of samples. 1 sample / 1 unique index)
172/ Mix for 1 sample:
10 μL 5X HF Phusion buffer
1 μL dNTPs (10 mM stock)
1 μL Oligo Forward NGS universal (1 μM stock)
1 μL NGS_universal_Forward_short (10 μM stock)
1 μL NGS_universal_Reverse_short (10 μM stock)
1 μL NGS_barcode_Reverse (1 μM stock)
2 μL library cDNA from mRNA reverse-transcription
1 μL Phusion polymerase
x μL H2O up to 50 μL
173/ Run the PCR program:
Initial denaturation 98°C 30”
Denaturation 98°C 10”
Annealing 42°C 10”
Elongation 72°C 10”
Hold 12°C ∞
Purification and analysis of RT-PCR products TIMING 2.5 hours
174/ For RT-PCR product purification using spin columns, please refer to the instruction manual provided by the manufacturer of the PCR Purification Kit
from Qiagen®, Macherey Nagel or Eurogentec.
- Elute in 20-30 μL H2O
175/ Measure concentration with the Nanodrop.
176/ Prepare a 9% TBE-acrylamide gel. Mix a volume of PCR product corresponding to 50 ng of DNA with x μL H2O to yield a combined volume of 5 μL. Add 1 μL 6X Loading Dye and load samples on the gel, along with 0.5 μL of Molecular weight ladder
177/ Gel electrophoresis conditions: 1 hour at 200 V in 1X TBE running buffer
178/ Staining: soak the gel for 15 minutes in SybrGold
CRITICAL STEP Do not use the same SybrGold solution more than 4 times. Prepare a fresh solution if the gel is not sufficiently stained.
! CAUTION Be sure to wear gloves and a lab coat if you do not want to stain your own DNA
179/ Imaging: Use Image Lab software to image your gel.
! CAUTION Acrylamide gels with or without SybrGold must be discarded in dedicated trash buckets for toxic waste.
PAUSE POINT Samples can be frozen at -20°C until the next step.
PREPARING THE SAMPLE MIX FOR NGS SEQUENCING
Measuring the concentration of individual library components with the Bioanalyzer TIMING 1 hour
180/ Assess the quality and quantity of individual DNA libraries for each sample/condition using a BioAnalyzer and a DNA 1000 chip. Please refer to the manufacturer’s recommendations and protocol
181/ Prepare a multiplexed library for NGS. Based on the DNA concentrations determined in Step 180, mix the individual library components according to the desired molar ratios
Determining the final library concentration with the Bioanalyzer prior to NGS TIMING 1 hour
182/ Assess the quality and quantity of the final library using a BioAnalyzer and a DNA 1000 chip. Place 1 μL of the final NGS library in several wells of the chip to obtain accurate concentrations for NGS. Please refer to the manufacturer’s recommendations and protocol
183/ Annotate your tube and wrap parafilm around the cap to prevent sample loss. Send your sample for next-generation sequencing