GSM2626698: Control-treated sample 12; Homo sapiens; OTHER
Cells were lysed using SDS in the presense of bisulfite. Bisulfite was dialysed out of the sample. Samples were treated with SDS and proteinase K to remove proteins. Nucleic acids were then extracted with phenol/chloroform. DNA was then digested with restriction enzymes and RNA-DNA hybrids were immunoprecipitated using S9.6 enzymes. DNA was sonicated using a covaris S-2 into 300 bp fragments. A Pico-Methyl Seq Library Prep Kit (Zymo Research, Irvine, California) was used to create the DNA sequencing library.  This kit is primarily designed for mapping 5-methylcytosine in small quantities of genomic DNA.  We omitted the bisulfite-treatment steps included in this kit.  Instead, only the library preparation steps were followed. These steps were followed for each sample as instructed by the manufacturer.  These steps amplified the DNA and used random priming to add indexes to the DNA library that are compatible with Illumina's TruSeq technology. As described in the Pico-Methyl Seq Library Prep Kit instructions, these indexes contain one of six barcodes that we used to differentiate samples added to the same lane during sequencing.