GSM1173120: E14-d6_hairpin-BS; Mus musculus; Bisulfite-Seq
Mouse ES cell genomic DNA was isolated using DNeasy Blood and Tissue kit (Qiagen, Frederick, MD). Total RNA was extracted with the miRNeasy extraction kit (Qiagen, Frederick, MD). Ten-microgram mouse genomic DNA was spiked with 0.01% unmethylated cl857 Sam7 Lambda DNA (Promega) and sonicated to 200bp fragments with Covaris S2 (AB). After purification (PureLink® PCR Purification Kit, Invitrogen), DNA fragments were then subjected to end repair with the end repair enzyme mix (NEB), dA tailing using Klenow 3’-5’ exo- (NEB) with purification at each step. Ligation to Biotin-modified hairpin adapter (P-CGCCGGCGGCAAG/iBiodT/GAAGCCGCCGGCGT) and Illumina TruSeq adapters were performed using T4 DNA ligase (NEB) overnight. Adapter-ligated DNA was digested with MseI and MluCI (NEB) for 1h at 37°C. After purification, DNA fragments were then pulled down using Dynabeads® MyOne™ Streptavidin C1 beads (Invitrogen). Bisulfite conversion was performed using the EpiTect Bisulphite Kit (Qiagen). After bisulfite conversion, the single-stranded uracil-containing DNA was subjected to 12 cycles of PCR reaction with Illumina TruSeq PCR primers (with specific index) and 2.5 U Pfu TurboCx Hotstart DNA polymerase (Agilent) to recover enough DNA for sequencing. After purification, size selection of 400-600 bp fragments was conducted with LabChip XT DNA Assay (Caliper) to yield longer sequences that are more amenable for unambiguous mapping to the reference sequence. RNA-seq libraries were constructed according to Illumima protocol and sequenced with the Illumina Hiseq 2000.