GSM1614734: CLL0625_RRBS; Homo sapiens; Bisulfite-Seq
Genomic DNA was extracted using Qiagen kit RRBS was performed according to a previously published protocol (Gu et al. 2010; Meissner et al. 2008) with minor modifications. For each sample, 1  g genomic DNA was first digested overnight using 20 units of MspI (New England Biolabs, NEB). The digested DNA was end-repaired and adenylated in a 20  l reaction consisting of 10U of Klenow fragments (exo-, Enzymatics, MA, USA), and 2  l premixed nucleotide triphosphates (1 mM dGTP, 10 mM dATP, 1 mM methylated dCTP). The reaction was incubated at 30 C for 30 min followed by 37 C for an additional 30 min. The methylated Illumina adapters were ligated with adenylated DNA fragments in a 20  l reaction containing 1  l concentrated T4 ligase (Enzymatics) at room temperature for 15 minutes. The ligation products were gel-selected for fragments with insertion sizes of 40 to 120 bp and 120 to 220 base pairs as previously suggested (Gu et al. 2010; Meissner et al. 2008).  Bisulfite treatment of the sequencing library was conducted using the EZ DNA methylation kit (Zymo Research, Irvine, CA) according to manufacturer's protocol. The final libraries were generated by PCR amplification of 5 ?m of bisulfite-converted template using PfuTurbo Cx Hotstart polymerase and Illumina pair-end PCR primers (PE1.0 and PE2.0).
GEO Accession
GSM1614734