GSM2676373: Dnmt3a null rep3; Mus musculus; Bisulfite-Seq
Spleen was harvested, and B220+IgD++GL7- B cells were sorted to a purity of 98-99%. Using the Trizol reagent, genomic DNA was extracted from the organic phase and further purified by performing Phenol-chloroform extractions. For each sample, 1500 ng intact genomic DNA and 1.5 ng unmethylated lambda DNA (Promega) were fragmented using a Covaris S220 Focused-ultrasonicator. Sequencing libraries were prepared using the NEXTflex Bisulfite-Seq Kit (BIOO Scientific) with a starting input of 1000 ng and using agarose gel size selection. Bisulfite conversion was performed using an EpiTect Bisulfite Kit (Qiagen). Bisulfite converted DNA product was PCR amplified for 10 cycles using KAPA HiFi Uracil+ Ready Mix (Kapa Biosystems). After library construction, all six libraries were equally multiplexed and sequenced using the Illumina NextSeq500 platform in paired-end format for 76 or 151 cycles at the NIEHS Epigenomics Core Laboratory.
GEO Accession
GSM2676373