GSM5694000: CTL_Adipose_BSseq_Rep1; Mus musculus; Bisulfite-Seq
For BS-seq, genomic DNA (1-2 µg) spiked with 0.5% unmethylated lambda genomic DNA (Promega) were sheared with a Diagenode Bioruptor to yield DNA fragments ranging from 200 to 500 bp. After purification of the fragmented DNA using phenol/chloroform, DNA fragments were end repaired, A-tailed, and ligated to methylated Illumina adaptors using as described for ChIP-seq. The EpiTect Bisulfite kit (QIAGEN, Cat. No. 59104) was used for bisulfite conversion and subsequent purification according to the manufacturer's instructions. Libraries for Illumina sequencing were constructed using the following standard protocol. The fragment end were repaired using the NEBNext End Repair Module, and adenosine was added at the 3' ends of fragment using Klenow fragment (3' to 5' exo minus, New England Biolabs). Precipitated DNA and input DNA were incubated with adaptors at room temperature for 1 hr with T4 DNA ligase (New England Biolabs) and amplified with Illumina primers.