GSM9885990: BSseq_XO_d2_r2; Mus musculus; Bisulfite-Seq
Whole Genome Bisulfite Sequencing (WGBS) for XO and XXΔXICB6 was performed in parallel, as described in (Mutzel et al, 2025). XXΔXICB6 data were published in (Mutzel et al, 2025). Briefly, XO B7 and XXΔXICB6 mES cells were differentiated for 0, 2 or 4 days and genomic DNA was extracted with the DNeasy Blood and Tissue Kit (Qiagen). Bisulfite-converted DNA libraries were prepared using the Accel-NGS Methyl-Seq DNA library kit (SWIFT BIOSCIENCES). Specifically, 200ng (in 50 μl lowTE) of purified DNA were fragmented to ~350 bp using the Covaris S2 system (10% duty cycle, intensity 5 for 2 × 45 s in Covaris AFA tubes) followed by a concentration step with Zymo columns (DNA Clean & Concentrator). 20 μl of DNA were used for bisulfite conversion overnight with the Zymo EZ-DNA methylation Gold Kit. Bisulfite-converted DNA was fully denatured by 2 min incubation at 95 °C and immediately transferred to ice. To anneal a truncated adapter, 15 μl of denatured DNA was mixed with 25 μl of Adaptase reaction mix (SWIFT) and incubated for 15 min at 37 °C, 2 min at 95 °C, and then cooled to 4 °C. Extension and second strand synthesis by using a primer complementary to the truncated adapter was performed mixing the Adaptase reaction mix with 44 μl of extension reaction mix (SWIFT) and incubation at 98 °C for 1 min, 62 °C for 2 min, 65 °C for 5 min and cooling to 4 °C. The samples were cleaned with 1.2 volumes of Ampure XP beads, 80% ethanol and eluted in 15 μl. Ligation of the second (truncated) adapter was performed at 25 °C for 15 min followed by an additional bead clean up with 1 volume of AmpureXP beads and 80% ethanol. Samples were individually indexed using unique dual indexed primer sets with 6 cycles of the following PCR program: 30s @ 98 °C, 6 cycles of 15s @ 98 °C, 30s @ 60 °C, 60s @ 68 °C, followed by a final 5 min incubation step at 68 °C. Libraries were cleaned up with 1 volume of Ampure XP beads. Quality was assessed using Agilent's Bioanalyzer and concentration was determined by qPCR. Libraries were pooled equimolarly and sequenced on a NovaSeq 6000 S2 flowcell (Illumina) in paired end 150 mode to yield ~ 120-278mio fragments.