When preparing single cell libraries, work in a dedicated pre-PCR environment, preferably a laminar flow cabinet. Treat surfaces and laboratory equipment with RNaseZap and wipe clean with 70% ethanol. Use dedicated stock solutions of all reagents and treat reagents and plastic ware with UV for 1 hour wherever appropriate.
Modification of scTEM-seq for low-input samples
The scTEM-seq protocol can be adapted to low-input samples such as single colonies or organoids. We have performed single-colony analysis by modifying the following steps:
o Single colonies were manually picked into 20μL RLT Plus Lysis Buffer containing 1U/µL SUPERase-In and stored at -80°C. 5μL of this lysate was used as input for the TEM-seq protocol.
o The TEM-seq library preparation was performed with the following modifications:
- Step 13: Dispense 5µL beads in 5µL of each colony lysate in a new 96-well plate.
- Step 27: Decrease number of PCR cycles in cDNA amplification from 20-24 (single cells) to 15 (single colonies).
- Step 104: Decrease number of PCR cycles from 35 (single cells) to 29 (single colonies).
o To perform single colony TEM-seq without prior separation of mRNA, transfer 10μL of each lysate to a new 96 well plate and begin at step 82.
Sample collection
Collect single cells by FACS or manual picking in 2.5μL RLT Plus Lysis Buffer containing 1U/µL SUPERase-In. With each collection include negative control samples (lysis buffer only, no cell).
scTEM-seq without scRNA-seq
To perform scTEM-seq without prior separation of mRNA, add 7.5μL of water to each cell lysate and begin protocol from the bisulfite conversion stage (step 82.).
Prepare scTEM-seq index plate
Dilute all oligos (supplementary table 1) to 1μM. Add equal volumes of forward and reverse oligo pairs to each well of a 96 well plate according to the layout provided in supplementary table 2. This plate can be stored at -20°C and reused.
Prepare wash buffers for MyOne Streptavidin C1 Dynabeads
Solution A (0.1M NaOH, 0.05M NaCl), Solution B (0.1M NaCl), and 2x B&W buffer (2M NaCl, 10mM Tris-HCl, 1mM EDTA) can be prepared in bulk (eg. 50mL) and stored in a refrigerator for repeated use.
Separation of Full-Length mRNA and gDNA
Prepare oligo-dT beads:
1. Place 60μL of MyOne Streptavidin C1 Dynabeads in a clean tube.
2. Place the tube on a magnetic rack and wait for the solution to clear.
3. Aspirate supernatant and remove from magnet.
4. Wash with Solution A:
o Resuspend beads in 60μL Solution A and incubate for 2 min.
o Place the tube on a magnetic rack and wait for the solution to clear.
o Aspirate supernatant and remove from magnet.
o Repeat wash once.
5. Wash with Solution B (0.1M NaCl):
o Resuspend beads in 60μL Solution B and incubate for 2 min.
o Place the tube on a magnetic rack and wait for the solution to clear.
o Aspirate supernatant and remove from magnet.
o Repeat wash once.
6. Wash with 2x B&W buffer (2 M NaCl, 10 mM Tris-HCl, 1 mM EDTA):
o Resuspend beads in 60μL 2x B&W and incubate for 2 min.
o Place the tube on a magnetic rack and wait for the solution to clear.
o Aspirate supernatant and remove from magnet.
o Repeat wash once.
7. Resuspend beads in 60μL of 2x B&W buffer
o For a full 96-well plate add 60μL of oligo dT (100μM).
o For fewer than 96 samples, transfer an appropriate volume of resuspended beads to a new tube and add an equal volume of oligo dT (100μM).
8. Incubate 15 min with rotation or occasional inversion.
9. Place the tube on a magnetic rack and wait for the solution to clear.
10. Aspirate supernatant and remove from magnet.
11. Wash with 1x B&W buffer:
o Resuspend beads in 60μL 1x B&W buffer and incubate for 2 min.
o Place the tube on a magnetic rack and wait for the solution to clear.
o Aspirate supernatant and remove from magnet.
o Repeat wash once.
12. Resuspend in the following buffer, scaling down for lower sample numbers:
Component Volume required for 60μL oligo-dT beads
H20 435μL
SuperScript II buffer (5x) 110μL
SUPERase-In (20U/μL) 27.5μL
Capture mRNA and perform gDNA washes:
13. Dispense 5μL of beads to each sample.
14. Vortex for 2 min and incubate 18 min.
15. Prepare the wash buffer:
Component Volume for 96 Samples
H20 3000μL
SuperScript II buffer (5x) 860μL
DTT (100mM) 430μL
Tween 20 (100%) 22μL
SUPERase-In (20U/μL) 86μL
16. Place sample plate on magnetic rack and wait for the solution to clear.
17. For each sample, transfer lysate to a new 96-well plate (DNA plate).
18. Wash oligo-dT beads in each sample:
o Resuspend beads in 10μL wash buffer and vortex to mix.
o Spin down sample plate and place on the magnetic rack.
o Wait for the solution to clear and transfer supernatant to the DNA plate.
o Repeat wash three times (perform 4 washes in total).
19. Seal DNA plate, spin down and store at -20°C until required.
Preparation of Amplified cDNA
Perform reverse transcription
20. For each sample, resuspend oligo-dT beads in 10μL of reverse transcriptase buffer:
Component Volume/Sample (μL) x110 (μL)
H20 3.59 394.9
Superscript II first-strand buffer (5×) 2.00 220
DTT (100 mM) 0.5 55
Betaine (5 M) 2.00 220
MgCl2 (1 M) 0.06 6.6
TSO (100μM) 0.10 11
dNTPs (10mM) 1 110
SuperScript II (200 UμL−1) 0.50 55
SUPERase-In (20 UμL−1) 0.25 27.5
Total volume 10 1100
21. Seal plate and vortex to mix.
22. Spin down and place plate in thermal cycler.
23. Incubate as follows:
Temperature (°C) Time
42 60 min
50 30 min
60 10 min
Amplify cDNA
24. To each sample, add 12μL of the PCR reaction mix detailed below.
Component Volume/Sample (μL) X110 (μL)
KAPA HiFi HotStart ReadyMix (2×) 11 1210
IS PCR primers (10μM) 0.25 27.5
H2O 0.75 82.5
25. Seal plate and vortex to mix.
26. Spin down and place plate in thermal cycler.
27. Incubate as follows:
Step Temperature (°C) Time Repeats
a 98 3 min 1
b 98 20 s
c 67 15 s
d 72 6 min go to step b 24 times
e 72 5 min 1
f 4 Hold 1
Purification and quality control of amplified cDNA (in Post-PCR)
Working in a post-PCR environment, purify the amplified cDNA using AMPure XP beads
28. Resuspend AMPure XP beads thoroughly by vortexing.
29. To ~22μL of amplified cDNA add 13.2μL of AMPure XP beads.
30. Seal plate, vortex to mix, and spin down.
31. Place plate on magnet and allow solution to clear.
32. Remove solution and discard.
33. Wash with 80% ethanol:
o Add 100μL 80% ethanol to beads.
o Seal plate, vortex to mix, and spin down.
o Place plate on magnet and allow solution to clear.
o Remove solution and discard.
o Repeat wash once.
34. Allow AMPure XP beads to dry for 5 min at room temperature.
35. Dispense 15μL distilled water into each well and resuspend AMPure XP beads by pipetting and spin down briefly.
36. Incubate at room temperature for 10 min to elute amplified cDNA from beads.
37. Spin down to pellet beads.
38. Run several representative samples on Tapestation HSD5000 (or similar) to check fragment size distribution.
39. Store amplified cDNA plate at -20°C until required for single cell RNA sequencing library preparation.
Preparation of scRNA-seq Libraries
Preparation of scRNA-seq libraries uses Illumina Nextera XT DNA Library Preparation Kit. More information regarding the protocol can be found in the Illumina Kit Documentation and by referring to smart-seq2 protocol 10,11.
Preparation
40. Work in a post-PCR environment.
41. The input amount of cDNA is important for the success of scRNA-seq library preparation. Quantify amplified cDNA using Qubit high-sensitivity dsDNA reagents and dilute to 0.2ng/μL.
42. Indexes are mixed in pre-prepared 96-well plates. Each well in the plate contains a unique combination of i5 and i7 indexes. Equal volumes of i5 and i7 indexes are combined in the plate, and then diluted 1:5 by adding 3 volumes of water.
Tagmentation reaction
43. Thaw Nextera XT reagents on ice, invert or vortex to mix and spin down briefly.
44. Add the following volumes in the order listed to each well of a new 96-well PCR plate. Pipette to mix after each addition.
o TD (2μL)
o Amplified cDNA (1μL, ~0.2ng)
o ATM (1μL)
45. Spin briefly to collect liquid in bottom of plate.
46. Place on the pre-programmed thermal cycler and run the tagmentation program.
o 55°C for 5 minutes
o Hold at 10°C
47. When the sample reaches 10°C, immediately proceed to the next step to inactivate the transposome
48. Add 1μL NT to each well. Pipette to mix.
49. Spin briefly to collect liquid in bottom of plate.
Amplification
50. Incubate at room temperature for 5 minutes.
51. Add 1μL each of Nextera XT Index 1 (i7) and Index 2 (i5) to each sample as appropriate. A total of 2μL of Index volume is added to solution.
52. Add 3μL NPM to each sample. Pipette to mix.
53. Spin briefly to collect liquid in bottom of plate.
54. Place on the pre-programmed thermal cycler and run the following PCR program.
Step Temperature (°C) Time Repeats
a 72 3 min 1
b 95 30 s 1
c 95 10 s
d 55 30 s
e 72 30 s go to step c 12 times
f 72 5 min 1
g 10 Hold 1
Library purification
55. Pool 2µL of each library in a 1.5mL microcentrifuge tube.
56. Add 0.8x AMPure XP beads to each well (e.g., 153µL beads to 192µL pool).
57. Vortex to mix, and spin down.
58. Incubate at room temperature for 5 mins.
59. Place tube on magnet and allow solution to clear.
60. Remove solution and discard.
61. Wash with 80% ethanol:
o Add 200μL 80% ethanol to beads in 96-well DNA plate.
o Vortex to mix, and spin down.
o Place plate on magnet and allow solution to clear.
o Remove solution and discard.
o Repeat wash once.
62. Allow AMPure XP beads to dry for 5 min at room temperature.
63. Resuspend AMPure XP beads in 15-30μL distilled water.
64. Mix by pipetting and spin down briefly.
65. Incubate at room temperature for 10 min to elute DNA from beads.
66. Spin down to pellet beads.
67. Run sample on Tapestation HSD5000 (or similar) to check fragment size distribution.
68. Store libraries at −20°C.
Preparation of scTEM-seq Libraries
Purification of gDNA
69. Work in pre-PCR biosafety cabinet.
70. Resuspend AMPure XP beads thoroughly by vortexing.
71. To ~47.5µL of solution in each well of the DNA plate add 40µL of AMPure XP beads.
72. Seal plate, vortex to mix, and spin down.
73. Incubate for 10 mins
74. Place plate on magnet and allow solution to clear.
75. Remove solution and discard.
76. Wash with 80% ethanol:
o Add 100µL 80% ethanol to beads in 96-well DNA plate, do not mix.
o With plate still on magnet remove ethanol and discard.
o Repeat wash once.
77. Dispense 10µL distilled water into each well of the 96-well DNA plate.
78. Resuspend AMPure XP beads by pipetting and spin down briefly.
79. Incubate at room temperature for 10 min to elute DNA from beads.
80. Spin down to pellet beads.
81. Proceed immediately to bisulphite conversion, or store DNA plate at -20°C until required for single cell bisulphite sequencing library preparation.
Bisulphite conversion
82. Prepare a vial of CT Conversion Reagent by adding 7.9mL of M-Solubilization Buffer and 3mL of M-Dilution Buffer. Vortex until all particles are dissolved, heating to 50°C if necessary. Add 1.6mL of M-Reaction Buffer and vortex thoroughly. This volume is sufficient for 200 samples. Prepared CT Conversion Reagent can be stored at −20°C for up to 4 weeks with no loss of performance.
83. Add 65μL of prepared CT Conversion Reagent to each single cell lysate. Incubate the mixture as follows:
Temperature (°C) Time
98 8 min
65 180 min
4 Hold
84. Prepare MagBinding Beads in a 96-well deep-well plate, by adding 5μL of MagBinding Beads to 300μL of M-Binding Buffer for each sample.
85. Add 75μL of converted DNA sample to the MagBinding Beads and M-Binding Buffer mixture.
86. Rinse the wells of the bisulphite conversion plate with 75μL of this mixture to collect any remaining sample and combine with the MagBinding Beads and M-Binding Buffer mixture.
87. Seal plate, vortex to mix, and spin down.
88. Incubate the mixture at room temperature for 5 min to bind the DNA to the MagBinding Beads.
89. Place the plate on a magnet until solution clears, and then remove and discard the supernatant.
90. Remove from the magnet and add 200μL of M-wash buffer to the beads.
91. Seal plate, vortex to mix, and spin down.
92. Place the plate on a magnet until solution clears, and then remove the supernatant.
93. Remove from the magnet and add 100μL of M-Desulfonation Buffer.
94. Seal plate, vortex to mix, and spin down.
95. Incubate the plate at room temperature for 15 min.
96. Place the plate on a magnet until solution clears, and then remove and discard the supernatant.
97. Wash with M-wash buffer:
o Add 200μL M-wash buffer to beads in 96-well plate.
o Seal plate, vortex to mix, and spin down.
o Place plate on magnet and allow solution to clear.
o Remove solution and discard.
o Repeat wash once.
98. Transfer to a heating element at 55°C for 15 min to dry the MagBinding Beads and remove residual M-wash buffer.
TE-specific PCR
99. Whilst MagBinding beads are drying, combine the following components of the PCR mix:
Component Volume/Sample (μL) (x110) (μL)
KAPA HiFi Uracil+ master mix (2x) 7.5 825
Water 3 330
100. Add 10.5μL of the PCR mix above to a new 96-well plate.
101. To the same plate, add 4.5μL primers from corresponding wells of the pre-prepared indexed SINE Alu primer set (supplementary table 2).
102. Pipette 15μL of PCR mix to each well of the deep-well plate containing MagBinding beads. Pipette up and down to resuspend beads, then transfer to a new 96-well plate.
103. Vortex beads to resuspend and incubate for 5 min.
104. Incubate as follows:
Step Temperature (°C) Time Repeats
a 95 5 min 1
b 98 20 s
c 53 15 s
d 72 60 s to go step b 35 times
e 72 10 min 1
f 4 Hold 1
105. Place the plate on a magnet and transfer the supernatant to a new plate or tube strip.
106. Add a 1.2x volume of AMPure XP beads to the amplicons.
107. Incubate for 10 min at room temperature.
108. Place the plate on a magnet until the solution clears. Remove and discard the supernatant.
109. Wash with 80% ethanol:
o Add 100μL 80% ethanol to beads.
o Vortex to mix, and spin down.
o Place plate on magnet and allow solution to clear.
o Remove solution and discard.
o Repeat wash once.
110. Remove from magnet and dry the beads for 10 min at room temperature.
111. Add 15μL of water to each sample and mix by pipetting.
112. Incubate the mixture at room temperature for 10 min to elute DNA from beads.
113. Place the plate on the magnet to pellet beads.
114. Run several representative samples on Tapestation HSD1000 to check fragment size distribution.
115. Take 2μL from each sample for quantification with the Qubit HS dsDNA kit or similar.
116. Normalise all samples to the same concentration, then pool equal volume of all samples from the same plate to a microcentrifuge tube for further indexing.
Indexing PCR
117. Prepare indexing PCR mix described below for each sample in the pre-PCR hood.
Component Volume/pool (μL)
KAPA HiFi HotStart ReadyMix (2×) 14.5
NEBNext Indexes (2µL of i7 and of 2µL i5) 4
118. In post-PCR, add 11.5μL of pooled purified amplicons from step 116 into the indexing PCR reaction.
119. Incubate as follows:
Step Temperature (°C) Time Repeats
a 98 45 s 1
b 98 15 s
c 65 30 s
d 72 30 s go to step b 5 times
e 72 5 min 1
f 4 Hold 1
120. Add a 1.2x volume of AMPure XP beads to the pooled samples.
121. Incubate for 10 min at room temperature.
122. Place the mixture on a magnet until the solution clears. Remove and discard the supernatant.
123. Wash each sample with 80% ethanol:
o Add 100μL 80% ethanol to beads.
o Vortex to mix, and spin down.
o Place plate on magnet and allow solution to clear.
o Remove solution and discard.
o Repeat wash once.
124. Dry the beads for 10 min at room temperature.
125. Add 30μL of water and mix by pipetting.
126. Incubate the mixture at room temperature for 10 min.
127. Place the plate on the magnet to pellet beads and transfer eluate to a new tube or plate.
128. Run samples on Tapestation HSD1000 to check fragment size distribution.
129. Store libraries at −20°C before sequencing.
130. Check concentration of library pools before sequencing using Qubit HS dsDNA kit or similar. Library pools with different NEBNext index pairs can be normalised and combined prior to sequencing.
Sequencing of scSTEM-seq Libraries
scRNA-seq (SMARTseq2) and scTEM-seq libraries are sequenced separately. Sequencing with the adapters used in scTEM-seq libraries should be performed using Illumina platforms such as the MiSeq. We used 300 cycle kits to cover internal indexes and not lose sequence information in the amplicon. Loading concentrations of 6pM with 5% PhiX spike-in have worked well for us, with yields of ~90% Q30. For accurate estimation of genome wide DNA methylation levels, scTEM-seq libraries should be sequenced with at least 20000 reads per sample. Bootstrapping analysis has previously shown that deeper sequencing delivers only small improvements in accuracy 8. The sample information provided for Illumina sequencing should only include the NEBNext or Nextera sequencing indexes used, secondary indexes from the scTEM-seq primer set are used for a second round of deduplication after sequencing.
Data Processing and analysis
Here we provide a basic guide with crucial commands required. A more in-depth guide including example bash processing scripts/pipeline, a SINE Alu annotation file and custom R functions to obtain methylation levels from .cov files are available on Github 12.
131. After demultiplexing of sequencing libraries using illumina indexes, fastq files of scTEM-seq pools are obtained. Fastq reads will contain the secondary scTEM-seq indexes used to demultiplex pools into individual cell libraries. Secondary demultiplexing is performed using Cutadapt. No trimming should be performed before this point. Forward and reverse read index lists are provided (supplementary files 1 and 2) that list indexes from the scTEM-seq index plate, with respective column and row numbers for sample naming. These index lists are input to Cutadapt along with fastq reads 1 and 2 for each pool. Output should be two fastq files per sample (read 1 and read 2) named by index plate well. “cutadapt -e 0.15 --discard-untrimmed --no-indels -g file:barcodes_fwd.fasta -G file:barcodes_rev.fasta -o {name1}{name2}_input.1.fastq.gz -p {name1}{name2}_input.2.fastq.gz input_R1_001.fastq.gz input_R2_001.fastq.gz”.
132. After demultiplexing, General QC metrics including read length and basecall quality should be checked with software such as Fastqc. Read counts for all samples should be collated and compared to check indexing was executed as expected.
133. Trimming is performed with Trim Galore. For 150bp read lengths (used in Miseq kits), 10bp is removed from the 5’ and 3’ ends of both forward and reverse reads to ensure index sequences are removed. “trim_galore --clip_R1 10 --clip_R2 10 --three_prime_clip_R1 10 --three_prime_clip_R2 10 --paired input.file”
134. Despite the conserved TE sequences targeted, scTEM-seq reads effectively map to the genome. Genome mapping is performed using Bismark. Reads are aligned in paired end and non-directional mode. Output is a single .bam file for each sample. Unique mapping rate for scTEM-seq reads should be >60%. Bam files should be retained to check number of unique read sites and number of SINE Alu sites covered. The number of unique SINE Alu annotations covered is a useful QC metric to identify low quality samples. ‘bismark --genome_folder /genome/folder/ --non_directional -1 input_1.fq.gz -2 input_2.fq.gz’.
135. Bam files are then used for methylation extraction with the Bismark methylation extraction tool. Methylation extraction produces site specific methylation data that is easily readable for quantification of average methylation levels. Output is a .cov file for each sample. ‘bismark_methylation_extractor --paired-end --bedgraph input.bam’.