GSM8184985: RRBS_Rbbp5-FKBP_DMSO_rep3; Mus musculus; Bisulfite-Seq
DNA was extracted from cells using DNeasy Blood and Tissue kit (Qiagen), and concentrations were quantified with Qubit dsDNA HS assay kit. For each sample, 2ug genomic DNA was spiked with 2ng unmethylated Lambda DNA (Promega) to allow assessment of the bisulfite-conversion reaction efficiency before DNA was ingested for 5 hours at 37°C using a methylation-insensitive restriction enzyme, MspI. Library preparation was performed using NEXTFLEX Bisulfite-Seq kit (Bioo Scientific) with methylated adaptors (Bioo Scientific) and 1µg input DNA according to manufacturer's protocol. DNA was quantified again using Qubit dsDNA HS assay kit before bisulfite conversion was performed with EZ DNA Methylation-Lightning kit (Zymo Research). Bisulfite-converted DNA was PCR amplified and purified following the protocol of NEXTFLEX Bisulfite-Seq kit (Bioo Scientific). RRBS libraries were quantified with Qubit dsDNA HS assay kit, and size distributions were analyzed on TapeStation 2200 (Agilent Technologies). Libraries were first pooled based on molar concentrations and then combined with RNA-seq or CUT&RUN samples to compensate for the inherently low ATGC complexity of RBBS libraries. The final library pools were denatured, loaded onto an Illumina Nextseq 550 with SE75 high-output kits, and sequenced as paired-end 36/36 cycles.