GSM7898087: KO_NPC_WGBS_Rep1; Mus musculus; Bisulfite-Seq
2 million cultured cells were harvested, washed in PBS and lysed with NP40 lysis buffer (0.32 M sucrose, 5 mM CaCl2, 3 mM Mg(Ace)2, 0.1 mM EDTA, 10 mM Tris pH8, 0.1% NP40). Nuclei were collected with centrifugation, incubated with 5 μl of 10 mg/ml RNase A (Sigma R6513) in 500 μl PK lysis buffer (1 M Tris pH8, 50 mM EDTA, 2% SDS, 2 M NaCl) at 37 ℃ for 15 minutes, and then with 10 μl of 20 mg/ml proteinase K (Sigma P2308) at 52 ℃ overnight with rotation. The samples were then added with phenol/chloroform (1:1) (Solarbio P1021), mixed with vortex, and centrifugated with 15000 g at RT for 10 minutes. The supernatant was taken and precipitated with isopropanol. The precipitated genomic DNA (gDNA) was washed in 70% ethanol, air dried and dissolved in buffer EB (Qiagen 19086). The gDNA was spiked in with 1% of unmethylated lambda DNA (Promega D1521) for efficiency validation of bisulfite conversion. 500 ng of gDNA was taken and sonicated to ~250 bp with Covaris M220 ultrasonicator. The DNA fragments were end-repaired and A-tailed with NEBNext Ultra II End Repair/dA-Tailing Module (NEB E7546), and then ligated with NEBNext Multiplex Oligos for Illumina (Methylated Adaptor, Index Primers Set 1, NEB E7535). After size-selected to 300~400 bp with Ampure XP beads (Beckman A63881), DNA was bisulfite converted with EZ DNA methylation kit (Zymo Research D5001), PCR amplified with KAPA HiFi HotStart Uracil+ ReadyMix (Kapa Biosystems KK2801), and the library was finally size-selected to 300~500 bp with Ampure XP beads. After analysis with Agilent Tapestation 4200, the library was sent for deep sequencing on Illumina NovaSeq 6000 (paired-end, 150bp).