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Total DNA was extracted using QIAamp Fast DNA Tissue Kit (Qiagen, Dusseldorf, Germany) following the manufacturer's procedure. The quantity of DNA was measured by reading A260/280 ratios by spectrophotometer. When A260/280 ratios located range 1.8 to 2.0, DNA was available. The fragmented DNA samples by using sonication (for WGBS) or using MspI (NEB, USA, for RRBS) were subjected to bisulfite conversion. The Accel-NGS Methyl-Seq DNA Library Kit (Swift, MI, USA) was utilized for attaching adapters to single-stranded DNA fragments. Briefly, as protocol shown below, the Adaptase step is a highly efficient, proprietary reaction that simultaneously performs end repair, tailing of 3' ends, and ligation of the first truncated adapter complement to 3' ends. The Extension step is used to incorporate truncated adapter 1 by a primer extension reaction. The Ligation step is used to add the second truncated adapter to the bottom strand only. The Indexing PCR step increases yield and incorporates full length adapters. Bead-based SPRI clean-ups are used to remove both oligonucleotides and small fragments, as well as to change enzymatic buffer composition. Finally, we performed the pair-end 2x150bp sequencing on an illumina Hiseq 4000 platform housed in the LC Sciences. |
Bisulfite-Seq |
GENOMIC |
Hybrid Selection |
PAIRED
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