GSM1367123: 75A-2; Homo sapiens; Bisulfite-Seq
Genomic DNA from the tumor and corresponding adjacent tissues was prepared using the QIAamp DNA Blood Mini Kit(Qiagen) following the manufacturer’s instructions. Prior to the library construction, 3µg of genomic DNA from each sample was fragmented using a Covarias sonication system to mean sizes of approximately 200-300bp. After fragmentation, libraries were constructed according to the Illumina Paired-End protocol. Briefly, the purified, randomly fragmented DNA was treated with a mix of T4 DNA polymerase, Klenow fragments, T4 polynucleotide kinase and a nucleotide triphosphate mix to repair the ends by blunting and phosphorylation. The blunted DNA fragments were subsequently 3’-adenylated using the Klenow fragment (3’-5’exo) and ligated by T4 DNA ligase to adapters synthesized with 5’ –methyl-cytosine instead of cytosine. The adaptor-ligated library was purified using AMPure XP beads (Beckman Coulter Genomics). 500 ng of each library was hybridized to Agilent SureSelect Methyl-Seq biotinylated RNA baits (84 Mb) for 24 h at 65 C. Biotinylated target hybrids were captured on Dynal MyOne Streptavidin T1 (Invitrogen), and purified through MinElute PCR column (Qiagen). Bisulfite conversion of the purified captured library was performed using the EZ DNA Methylation Gold Kit (Zymo Research) as per manufacturer’s instructions. The bisulfite converted captured library was amplified by PCR with 14 PCR cycles, then purified by AMPure XP beads and quantified by Agilent 2100 system and qPCR. Methyl-Seq libraries were then sequenced on the HiSeq 2000 platform according to the manufacturer’s instructions, and 2 ×90bp paired-end reads were generated. Methyl-Seq libraries were prepared for sequencing using standard Illumina protocols.
GEO Accession
GSM1367123