title: GSM1180317: methylC-Seq in WT, biological rep 2, technical rep 1; Mus musculus; Bisulfite-Seq
library_construction_protocol: Genomic DNA extraction Purified genomic DNA was fragmented to an average size of 300bp with a Biorupter 300 (high power, 15s on, 15s off, 40 cycles). Spike in unmethylated lambda DNA control was added prior to sonication at 0.5% of total genomic DNA. DNA was end-repaired, adenylated, and ligated to methylated (5mC) adapters (Illumina) according to standard Illumina protocols for genomic DNA library construction, maintaining the proper molar ratios of adapter to insert. Adapter ligated fragments of size 200-650bp were gel purified by 2% agarose gel electrophoresis and sodium-bisulfite treated using the MethylCode kit (Invitrogen). Bisulfite treated adapter-ligated DNA was amplified by PCR with the following conditions in a 50uL final reaction volume:  2.5U PfuTurbo Cx Hotstart DNA polymerase, 5uL 10X PfuTurbo Cx reaction buffer, 0.75uL 20mM dNTPs, 5uL Illumina PCR Primers. Cycling parameters: 95ºC 5min, 98ºC 30 sec, followed by 8-11 cycles of 98ºC 15 sec, 60ºC 30 sec, 72ºC 4 min, and ending with 72ºC 10 min. The number of PCR cycles used was determined by quantification of bisulfite treated adapter-ligated DNA by qPCR (KAPABiosystems library quant kit for Illumina libraries) such that the final library concentration obtained was approximately 20nM. Final sequencing libraries were purified by 2% agarose gel electrophoresis and quantified by qPCR (KAPABiosystems library quant kit for Illumina libraries).