GSM2088209: N37-SI; Homo sapiens; Bisulfite-Seq
Approximately 200 ng of genomic DNA of primary tissues (N37) obtained from the Li Lab was sheared into an average size of 400 bp in a Covaris micro TUBE with Covaris E210 ultrasonicator. Fragmented genomic DNA was constructed into Illumina paired-end sequencing libraries using KAPA Library Preparation kit (KAPA Biosystems) following manufacturer’s instruction with modifications. After end-repair and dA-tailing, ligation with methylated adapters was performed at 20 ˚C for 15 min in the presence of 10-fold molar excess of Illumina methylated adapters (Illumina). The ligation mixture was purified with an equal volume of Agencourt AMPure XP beads (Beckman Coulter) and eluted with 23 µL of 10mM Tris-HCl, pH8.5. Next, 20 µL of adaptor ligated DNA was bisulfite converted using EZ DNA Methylation-Lightning kit (Zymoresearch) following manufacturer’s protocol and eluted with 30 µL of 10mM Tris-HCl, pH8.5. Bisulfite converted DNAs were amplified using iQ SYBR Green Supermix (Bio-Rad) with 200 nM each of PCR primer PE1.0 and multiplexing PCR primer for 10 cycles in 100 µL total volume. PCR products were purified with 0.8X volume of Agencourt AMPure XP beads (Beckman Coulter) and eluted with 50 µL of 10mM Tris-HCl, pH8.5, pooled in equimolar ratios, and size selected using 6% TBE gels for 400-600 bp. The concentration of sequencing libraries was quantified by qPCR using KAPA Library Quantification kit (KAPA Biosystems). Libraries were sequenced on both GAIIx for SE 36 cycles and HiSeq Run for PE 100 cycles.
GEO Accession
GSM2088209