title: GSM1608225: sample_517 (validation); Homo sapiens; Bisulfite-Seq
library_construction_protocol: Genomic DNA was extracted using the QIAamp DNA Blood Mini Kit (Qiagen). 1µg of genomic DNA was fragmented into approximately 200-300bp using a Covarias sonication system (Covarias). After purification, the fragmented fractions were treated with a mix of T4 DNA polymerase, Klenow Fragment and T4 polynucleotide kinase to repair blunt and phosphorylate ends. Reduced representation bisulfite sequencing (RRBS) libraries with single MspI digestion  were constructed for all samples. 1.5 µg of genomic DNA was digested with 500U of MspI  enzymes (NEB) in 100µl reactions at 37°C for 16h. After that MspI was inactivated at 65°C for  20 min. After digestion the Ilumina Paired-End protocol was used to construct the libraries  including end-repair, dA addition and methylated-adaptor ligation. MspI-digested DNA was  purified and size-selected on a 2% agarose gel. To obtain DNA fragments of 40-120 bp and  120-220 bp of length, adaptor-ligated fragments of 160-240 bp and 240-340 bp were excised,  respectively. Subsequently bisulfite conversion was conducted by ZYMO EZ DNA  Methylation-Gold KitTM (ZYMO) following the manufacturer’s instructions. The final libraries  were generated by PCR in a final reaction volume of 50 µl, comprising 20 µl purified DNA, 4  µl 2.5 mM dNTP, 1 µl PCR primer PE 1.0, 1 µl Index primer, 5 µl 10×buffer, 0.5 µl JumpStartTM  TaqDNA polymerase and 28.5 µl UltraPureTM Water. The PCR program was as follows: 94 ◦C  for 60 s, 13/15 cycles (13 cycles for 160–240 bp, 15 cycles for 240–340 bp) of 94 ◦C for 30 s,  58 ◦C for 30 s and 72 ◦C for 30 s. finally 72 ◦C for 5 min and products were then hold at 12 ◦C.  Between each step in the above mentioned process the DNA was purified using MinElute og  QIAquick columns according to protocols from Qiagen. Libraries were then subjected to  paired-end multiplex sequencing on Hiseq 2000 platform after quality control.
attributes.tag: GEO Accession
attributes.value: GSM1608225