GSM8464528: HCT116, Valine deprivation, MAB-seq; Homo sapiens; OTHER
500 ng of DNA extracted from HCT116 cells with or without the treatment of valine deprivation was first spiked-in with unmethylated lambda DNA (1:500), and break the DNA to 600-700 bp. The broken DNA was treated by M.SssI (New England Biolabs, M0226M) in a 50 μL reaction. In the treatment, DNA was first incubated with 1.0 unit/μl M.SssI methylase (New England Biolabs, M0226M) for 4 h in 50 μL reaction (1 μL of 4 U/μL M.SssI, 5 μL of 1.6 mM SAM, 5 μL of 10×Methyltransferase Reaction Buffer, add ddH2O up to 50 μL). DNA was purified by using DNA Clean & Concentrator kit (Zymo，D4013). M.SssI-treated DNA was then subjected to bisulfite conversion using EZ DNA Methylation-Direct kit (Zymo，D5021). Bisulfite-treated DNA were purified and subjected to library construction with EpiArt DNA Methylation Library Kit (Vazyme, NE103) and VAHTS Multiplex Oligos Set 4 (Vazyme, N321) according to the instruction manual (Illumina). Briefly, the single-stranded heat-denatured DNA fragments were ligated to the 3' adapter ligation and 5' adapter ligation. Next, the ligated DNA was extended to the second strand synthesis and the methylated adapters were ligated. The DNA was ampliﬁed for 10 cycles using Illumina 8-bp dual index primers. Finally, the MAB-seq libraries were analyzed by Agilent 2100 Bioanalyzer and sequenced on an Illumina NovaSeq 6000 platform by 150 bp paired-end sequencing.