GSM2425092: SSEA4+_SSCs_WGBS_rep1; Homo sapiens; Bisulfite-Seq
Genomic DNA was extracted from pooled SSEA4+ germline stem cells using AllPrep RNA/DNA/Protein Mini Kit (QIAGEN cat # 80004). Library construction was performed using the Epicentre EpiGnome Methyl-Seq Kit (EGMsK89312) as described below. Briefly, genomic DNA (50-100 ng) was denatured and bisulfite converted using the Zymo Research EZ DNA Methylation-Gold Kit (cat# D5005) in a reaction containing 0.5 ng of unmethylated lambda DNA (Promega cat# D1521) as a control.  Following purification, the bisulfite converted DNA was hybridized with oligonucleotides consisting of random hexamers linked to Illumina P5 adapter sequence and strand replication was accomplished using EpiGnome polymerase.  Double-stranded DNA was heat denatured to enable ligation of the EpiGnome Terminal Tagging Oligo which adds Illumina P7 adapter sequence to the 3% 2019 end of the replicated strand.  Adapter-ligated DNA molecules were enriched by 10 cycles of pcr and the amplified library was subsequently purified using Agencourt AMPure XP beads (Beckman Coulter Genomics cat#A63881).  The concentration of the library was measured using the Qubit dsDNA HS Assay (Invitrogen cat#Q32854) and an aliquot of the library is resolved on an Agilent 2200 Tape Station using a D1000 (cat# 5067-5582 and 5067-5583) or a High Sensitivity D10000 (cat# 5067-5584 and 5067-5587) assay to define the size distribution of the sequencing library.  Libraries were adjusted to a concentration of approximately 10 nM and quantitative PCR was performed using the Kapa Biosystems Kapa Library Quant Kit (cat# KK4824) to calculate the molarity of adapter-ligated DNA molecules.  The concentration was further adjusted following qPCR to prepare the library for Illumina sequence analysis. Sequencing libraries were then sequenced on a 125-cycle paired-end run on an Illumina HiSeq 2500 instrument.
GEO Accession
GSM2425092