GSM8084423: HCT116_Cl9_shU1_Recovery_D28; Homo sapiens; Bisulfite-Seq
Cells were lysed overnight at 37°C in 2 mls of TE-SDS buffer (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA, 0.5% SDS), supplemented with 100 μl of 20 mg/ml proteinase K. DNA was purified by phenol:chloroform extraction in three phases: (1) 100% phenol, (2) phenol:chloroform:isoamyl alcohol (25:24:1), and (3) chloroform:isoamyl alcohol (24:1). For each phase, the aqueous layer was combined with the organic layer in a 1:1 ratio. Samples were quickly shaken, allowed to sit on ice for approximately 5 minutes, and then separated by centrifugation at 1,693 RCF for 5 minutes at 4°C. The top aqueous layer was then transferred to a new tube for the next organic phase. Following extraction, DNA was precipitated with 1/10 volume 3 M sodium acetate pH 4.8 and 2.5 volumes 100% ethanol and stored overnight at -20°C. Precipitated DNA was pelleted by centrifugation at 17,090 RCF for 30 minutes at 4°C. The pelleted DNA was washed twice with 70% ethanol, allowed to dry for 15 minutes, and resuspended in TE buffer (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA). Samples were then treated with 1 mg/ml RNAse A at 37°C for 30 minutes and then re-purified by ethanol precipitation as described. 200 ng of DNA was bisfulfite converted using the EZ DNA Methylation-Gold Kit (Zymo, #D5005) according to manufacturer's instructions. Input DNA was spiked with unmethylated lambda DNA (0.5%) (Promega, #D1521). Replicate bisulfite libraries were generated with the CEGX TrueMethyl® Whole Genome Kit (CEGX, #CEGXTMWG, v3.1) according to manufacturer's instructions. Libraries were sequenced on the Illumina X Ten. Sequencing reads from WGBS data were aligned to the human genome using v1.2 of an internally developed pipeline Meth10X41. This is publicly available and can be downloaded from https://github.com/luuloi/Meth10X.