GSM5918614: E14 mESC BL_H-H-Dyad-seq; Mus musculus; OTHER
GSM5918614
Bulk genomic DNA was extracted using the DNeasy kit (Qiagen, 69504) according to the manufacturer's recommendations. Bulk total RNA was extracted using TRIzol reagent (Invitrogen, 15596018) according to the manufacturer's recommendations. For single cell experiments, whole cell lysate was used by soring cells via FACs directly into lysis buffer. For all bulk Dyad-seq experiments, 100 ng of purified genomic DNA was resuspended in 20 µL of glucosylation mix (1x CutSmart buffer (NEB, B7204S), 2.5x UDP-glucose, 10 U T4-BGT (NEB, M0357L)) and the samples were incubated at 37°C for 16 hours. Afterwards, 10 µL of protease mix (100 µg protease (Qiagen, 19155), 1x CutSmart buffer) was added to each sample, and the samples were heated to 50°C for 5 hours, 75°C for 20 minutes, and 80°C for 5 minutes. After this, processing of the samples differed based on the version of Dyad-seq used. For M-M-Dyad-seq and M-H-Dyad-seq, 10 µL of MspJI digestion mix (2 U MspJI, 1x enzyme activator solution, 1x CutSmart buffer) was added to each sample and the samples were heated to 37°C for 5 hours, and 65°C for 20 minutes. Next, 1 µL of barcoded 1 µM double-stranded adapter was added. Then 9 µL of ligation mix (1.11x T4 ligase reaction buffer, 4.44 mM ATP (NEB, P0756L), 2000 U T4 DNA ligase (NEB, M0202M)) was added to each sample, and the samples were incubated at 16°C for 16 hours. For H-M-Dyad-seq and H-H-Dyad-seq, 10 µL of AbaSI digestion mix (10 U AbaSI (NEB, R0665S), 1x CutSmart buffer) was added to each sample and the samples were incubated at 25°C for 2 hours, and then heated to 65°C for 20 minutes. Next, 1 µL of barcoded 1 µM double-stranded adapter was added. Then 9 µL of ligation mix (1.11x T4 ligase reaction buffer, 4.44 mM ATP (NEB, P0756L), 2000 U T4 DNA ligase (NEB, M0202M)) was added to each sample, and the samples were incubated at 16°C for 16 hours. After ligation, up to three barcoded libraries of the same type were pooled and all Dyad-seq versions were subjected to a 1x AMPure XP bead cleanup (Beckman Coulter, A63881), and eluted in 40 µL of water. M-M-Dyad-seq and H-M-Dyad-seq samples were then concentrated to a volume of 28 µL and subjected to nucleobase conversion using the NEBNext enzymatic methyl-seq conversion module (NEB, E7125S) according to the manufacturer's recommendations except for performing the final elution step in 40 µL of water. For M-H-Dyad-seq and H-H-Dyad-seq samples, nucleobase conversion was performed using the NEBNext enzymatic methyl-seq conversion module similar to what has been described previously11. Briefly, samples were first concentrated to a volume of 17 µL. Then 4 µL of formamide (Sigma-Aldrich, F9037-100ML) was added and the samples were heated to 85°C for 10 minutes before being quenched on ice. APOBEC nucleobase conversion was performed as described by the manufacturer except for two minor changes. Samples were incubated at 37°C for 16 hours, and the final elution step was performed using 40 µL of water. To all Dyad-seq versions, the nucleobase converted samples were subjected to one round of linear amplification. To do this, 9 µL of amplification mix was added (5.56x NEBuffer 2.1 (NEB, B7202S), 2.22 mM dNTPs (NEB, N0447L), and 2.22 uM Linear amplification 9-mer: 5'- GCCTTGGCACCCGAGAATTCCANNNNNNNNN -3') and the samples were heated to 95°C for 45 seconds before being quenched on ice. Once cold, 100 U of high concentration Klenow DNA polymerase (3'-5' Exo-) (fisher scientific, 50-305-912) was added. Then samples were quickly vortexed, centrifuged and then incubated at 4°C for 5 minutes, followed by an increase of 1°C every 15 seconds at a ramp rate of 0.1°C per second till the samples reach 37°C which was then held for an additional 1.5 hours. Afterwards a 1.1x AMPure XP bead cleanup was performed, and the samplers were eluted in 40 µL of water before being concentrated down to 10 µL. The entire sample was then used in a linear PCR reaction by adding 15 µL of PCR mix (1.67x high-fidelity PCR mix (NEB, M0541L) and 0.67 µM Extended RPI primer: 5'- AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGACGATCGGTGTAGTGGGTTTGG-3') and performing PCR as follows: Initial denaturing at 98°C for 30 seconds, followed by 16 cycles of 98°C for 10 seconds, 59°C for 30 seconds, and 72°C for 30 seconds, and a final extension step at 72°C for 1 minute. Next, 5 µL of the linear PCR product was amplified further in a standard Illumina library PCR reaction, incorporating a uniquely indexed i7 primer. The remaining linear PCR product was stored at -20°C. To the final sequencing library, two 0.825x AMPure XP bead cleanups were performed with a final elution volume of 15 µL in water. The libraries were then quantified on an Agilent Bioanalyzer and Qubit fluorometer. Finally, libraries were subjected to paired-end 150 bp Illumina sequencing on a HiSeq platform.