title: GSM5050891: MethylC-seq_P0_Control_rep1; Mus musculus; Bisulfite-Seq
library_construction_protocol: The frontal cortex tissue was rapidly dissected from 1-2 adult mice on ice, and the INTACT method was used to affinity purify GFP+ nuclei. Isolated nuclei were resuspended in PBS for DNA purification using the DNeasy Blood and Tissue kit (Qiagen 69504) following the standard kit protocol. MethylC-seq libraries were constructed as previously described [Urich M. et al. MethylC-seq library preparation for base-resolution whole-genome bisulfite sequencing. Nature Protocols. 2015 Mar;10(3):475-83.]. All DNA obtained from the extraction was spiked with 0.5% unmethylated Lambda DNA. The DNA was fragmented with a Covaris S2 (Covaris, Woburn, MA) to 200 bp, followed by end repair and addition of a 3' A base. Cytosine-methylated adapters provided by Illumina (Illumina, San Diego, CA) were ligated to the sonicated DNA at 16˚C for 16 hours with T4 DNA ligase (New England Biolabs). Adapter-ligated DNA was isolated by two rounds of purification with AMPure XP beads (Beckman Coulter Genomics, Danvers, MA). Adapter-ligated DNA (≤450 ng) was subjected to sodium bisulfite conversion using the MethylCode kit (Life Technologies, Carlsbad, CA) as per manufacturer's instructions. The bisulfite-converted, adapter-ligated DNA molecules were enriched by 8 cycles of PCR with the following reaction composition: 25 µL of Kapa HiFi Hotstart Uracil+ Readymix (Kapa Biosystems, Woburn, MA) and 5 µl TruSeq PCR Primer Mix (Illumina) (50 µl final). The thermocycling parameters were: 95˚C 2 min, 98˚C 30 sec, then 8 cycles of 98˚C 15 sec, 60˚C 30 sec and 72˚C 4 min, ending with one 72˚C 10 min step. The reaction products were purified using AMPure XP beads. Up to two separate PCR reactions were performed on subsets of the adapter-ligated, bisulfite-converted DNA, yielding up to two independent libraries from the same biological sample.