DPC-Seq sample processing
1. Seed 160,000 cells per well in 6-well plates in 10% FBS media.
2. After 24 h, wash with 1x PBS and add 1% FBS media.
3. After 24 h, change media to 1% FBS media with 1.75 mM formaldehyde (Methanol-free).
4. After 1 h, remove media, wash twice with 1x PBS.
5. Remove all PBS using 10 µl pipette tip on vacuum pump.
6. Lyse the cells with 150 µl SDS-buffer (20 mM Tris pH 7.5/HCl, 2% SDS).
7. Harvest cells by scraping and directly freeze at -80°C.
8. Next day, thaw samples only directly before sonicating by shaking at 1200 rpm at 55°C.
9. Transfer samples to 130 µl Covaris pre-slit snap-cap microTUBEs.
10. Sonicate with a peak power of 105 Watts for two minutes.
11. Transfer samples to fresh 1.5 ml tubes and add 270 µl SDS-buffer.
12. Take 40 µl as input control.
13. Add 400 µl KCl-buffer (20 mM Tris pH 7.5/HCl, 200 mM KCl), invert to mix content and incubate on ice for 5 min.
14. Centrifuge the samples for 5 min at 16,100xg at 4°C.
15. Take 600 µl of supernatant as soluble DNA and remove the remaining buffer.
16. Add 400 µl KCl-buffer, shake at 1200 rpm at 55°C for 5 min and incubate on ice for 5 min.
17. Centrifuge the samples for 5 min at 16,100xg at 4°C and discard supernatant.
18. Repeat step 16 and 17 twice more.
19. Add 400 µl KCl-buffer containing Proteinase K (0.2 mg/ml), shake at 1200 rpm at 55°C for 5 min and incubate at 55°C for 40 min without shaking.
20. Add 10 µl Ultrapure BSA (50 mg/ml), invert to mix content and incubate on ice for 5 min.
21. Centrifuge the samples for 5 min at 16,100xg at 4°C.
22. Take 400 µl of supernatant as crosslinked DNA and discard the pellet.
23. Add 3 and 2 µl RNAse A (DNAse and Protease-free) to each soluble and crosslinked DNA respectively, invert to mix content and incubate at 37°C for 30 min.
24. Mix Qubit dye in Qubit buffer (Qubit dsDNA HS assay) diluted 1 in 200.
25. Pipette 10 µl of all samples in technical duplicate in a black 96-well plate.
26. Add 190 µl of Qubit mix to each well using multichannel-pipette.
27. Measure the fluorescence of samples as indicated in Qubit protocol.
DPC-Seq DNA library preparation
1. Combine technical replicates at equimolar ratios for a minimum total of 50-500ng of DNA.
2. Add sodium acetate pH 5.2 to a final concentration of 300mM, 1µl of glycogen and 2.5x of the resulting volume of ethanol.
3. Incubate at -20°C overnight to precipitate the DNA.
4. Spin DNA samples at 21,000xg for 30 min at 4°C to pellet DNA.
5. Aspirate supernatant and add 1ml of 80% ethanol, spin at 21,000xg for a further 20 min.
6. Aspirate supernatant and air dry pellet until all ethanol has evaporated.
7. Resuspend pellet in 20µl of nuclease free water.
8. Cast 1% agarose TAE gels in 1.5mm Mini-PROTEAN cassettes (Bio-Rad).
9. Add 5µl of 5x loading buffer containing bromophenol blue to DNA samples.
10. Dilute DNA ladder of preference 1 in 100 in 1x DNA loading buffer.
11. Load ladder and DNA samples into the gel ensuring a minimum space of 1 empty well between each loaded well, as sample/ladder can commonly crossover to adjacent wells.
12. Run the gel at 100V until the bromophenol blue band has reached 2/3 of the way down the gel.
13. Remove the gel from the cassettes and cut of the wells and discard them as they will break off during washes.
14. Place the gel in a container containing 1x TAE with SYBR-Gold (Invitrogen) diluted 1 in 10,000.
15. Gently rock the gel for 5 min, then transfer the gel to a fresh container with 1x TAE.
16. Rock for 5 minutes, replace the buffer with fresh 1x TAE.
17. Place the gel on a blue light transilluminator to visualise the DNA, DPC DNA should be a smear from ~400-1000bp, though this can vary heavily with sonication conditions.
18. Using a fresh scalpel for each sample, excise the DNA smear, ensuring any high-molecular weight DNA bands (usually above the highest ladder band) are not included.
19. Slice excised gel into smaller pieces (usually 5-10 pieces depending on slice size) and places the pieces into a fresh DNA LoBind tube using the scalpel to manipulate the gel.
20. To each tube add 500µl of gel-extraction buffer (10mM Tris pH 8.0, 1mM EDTA, 0.02% SDS) and rotate at 4°C overnight.
21. Transfer gel and buffer to Spin-X columns with 0.22µm cellulose acetate membranes (Costar, CLS8160) and spin at 14,000xg for 10 min at 4°C.
22. Transfer the eluted buffer to a fresh DNA LoBind tube and add sodium acetate pH 5.2 to a final concentration of 300mM, 1µl of glycogen and 2.5x of the resulting volume of ethanol.
23. Incubate at -20°C overnight to precipitate the DNA.
24. Spin DNA samples at 21,000xg for 30 min at 4°C to pellet DNA.
25. Aspirate supernatant and add 1ml of 80% ethanol, spin at 21,000xg for a further 20 min.
26. Aspirate supernatant and air dry pellet until all ethanol has evaporated.
27. Resuspend pellet in 60µl of nuclease free water.
28. Mix Qubit dye in Qubit buffer (Qubit dsDNA HS assay) in 1:200 ratio.
29. Pipette 5µl of all samples in technical duplicate in a black 96-well plate.
30. Add 190µl of Qubit mix to each well using multichannel-pipette.
31. Measure the fluorescence of samples as indicated in Qubit protocol.
32. Dilute a minimum of 10ng of DNA into 50µl of nuclease free water and store the remaining sample in -80°C.
33. The 10ng DNA samples are then suitable for final end-repair, adapter ligation and PCR based DNA library preparation kit.
34. We used the NEBNext Ultra-II kit (NEB E7645L) according to the manufacturer´s instructions using a 1 in 10 adapter, no size selection and dilution and 7 PCR cycles.
35. Library concentration and quality can then be assessed by Tapestation, Bioanalyzer or agarose gel and concentration can be accurately determined by Qubit assay or qPCR.
36. Sequencing should always be conducted in paired end, with a minimum of 35 cycles per read, though we recommend PE50.
DPC-Seq analysis
1. Fastq files are first filtered for high quality reads using fastp (v0.23.2) with default quality settings and minimum length of 5. “-g” is also specified to ensure trimming of poly-G tracts inappropriately called at the end of reads by Illumina 2-color chemistry based sequencers.
2. Bowtie2 is used to align reads to the appropriate reference genome, specifying correct orientation of reads with ‘--fr’, a maximum insert size tailored to your expected maximum based on the gel smear from step 17 using ‘--maxins’ and allowing dovetailing with ‘--dovetail’.
3. Sort and index the alignments using samtools.
4. For the generation of metagenes, bamCoverage from Deeptools (v3.5.0) is used to generate BigWig files which can then be used with plotHeatmap and plotProfile from Deeptools to generate the desired plots.
5. For per gene read coverage, bedtools (v2.30.0) coverage is used to convert alignments to BED files with bamToBed and then to calculate coverage at every gene in the Gencode human genome annotation (GRCh38.p14) with coverage.
6. These coverage files are then normalised to total number of aligned reads.
7. The coverage files can then be loaded into R for statistical analysis and plot generation.