GSM9688056: 51264_1625168B_young-female-B-cells-second-sorting; Mus musculus; Bisulfite-Seq
Mouse genomic DNA and RNA were extracted from sorted MPAL or lineage control cells following the direction of the AllPrep DNA/RNA Mini kit (Qiagen, #80204) and stored at -80°C until the sequencing. 250 ng of genomic DNA was digested with MspI at 37°C overnight. In the same reaction tube, ends of the fragments were filled in and a 3′ adenosine was added with Klenow Fragment (3′→5′ exo-minus), and followed by the ligation reaction to add on the methylated universal adaptors. Insert fragments over 40 bp were purified with 1.6x Ampure XP beads. Bisulfite conversion was done with EZ DNA Methylation kit (Zymo Research, Cat D5001) and the final library was amplified with Pfu Turbo Cx Hotstart DNA Polymerase (Agilent, Cat 600414). The final library size of 160-500bp was purified with 6% of TBE polyacrylamide gels, quantified by Qpcr and sequenced on Illumina NovaSeq6000 S4 flow cell.