title: GSM2309191: WGBS PreC_2; Homo sapiens; Bisulfite-Seq
library_construction_protocol: Genomic DNA for both cell lines was extracted using QIAamp DNA Mini and Blood Mini kit following the manufacturer’s protocol for cultured cells (Qiagen). Whole genome bisulfite sequencing libraries were prepared using the Illumina Paired-end DNA Sample Prep Kit (Illumina). Briefly, DNA (1ug), was spiked with 0.5% unmethylated lambda DNA (Promega) in a final volume of 50-65ul.  DNA was sheared to 150-300bp by sonication with a Covaris S2. Library preparation was performed according to the manufacturer’s protocol - fragments were end-repaired and adenylated before ligation of Illumina Truseq adaptors.  Gel size selection (260-330bp) was used to purify and size select the ligated DNA, using Qiagen Gel extraction kit (Qiagen, part #28704) and DNA was eluted in 20ul H2O.  Bisulphite treatment was then carried out as previously described [PMID: 17406479], with the bisuphite reaction being performed for 4 hrs at 55°C. After bisulphite cleanup the DNA pellet was resuspended in 50 ul H2O. The adaptor-ligated bisulfite-treated DNA was enriched by performing 5 independent PCR reactions for 10 cycles using PfuTurboCx Hotstart DNA polymerase (Stratagene) in a volume of 50ul per PCR reaction.  The 5 independent PCRs were pooled together, cleaned up using the MinElute PCR purification kit and eluted in 20ul Qiagen EB buffer. Library quality was assessed with the Agilent 2100 Bioanalyzer using the High-sensitivity DNA kit (Agilent, CA, USA). DNA was quantified using the KAPA Library Quantification kit by quantitative PCR (KAPA Biosystems). Paired-end 100bp sequencing was performed for each library on the Illumina HiSeq 2500 platform using Truseq v3 cluster kits and SBS kits.
attributes.tag: GEO Accession
attributes.value: GSM2309191