Irs1KO_1
Genomic DNA of 5  AL, 5 DR, 4 WT and 4 Irs1KO animals  was isolated used as input for BS-Seq library generation.Total nucleic acid  isolated from the 2i release experiment underwent bisulfite conversion using  Zymo reagents according to manufacturere's protocol.KAPA HiFi Uracil+ Master  Mix (Kapa Biosystems) was used to amplify regions of interest using 30nM  primers), with the reverse primer including an 6 bp sample barcoe. The PCR  program was: 95C 5min; 35 repeats of 98C 20s, 60C 15s, 72C 60s; 72C 10min.  Amplicons were pooled for each sample and purified using Ampure XP beads  (Agencourt), before a second round of PCR was used to incorporate Illumina  Adaptor sequences and index samples. The PCR reaction included 11ul pooled  amplicons, 200nM indexed PE1.0 and iPCRTag primers, and KAPA HiFi Master Mix  (Kapa Biosystems). The PCR program was: 98C 45s; 5 repeats of 98C 15s, 65C  30s, 72C 30s; 72C 5min. Samples were then pooled and purified before library  QC and sequencing was performed with up to 144 samples included on a 150bp  paired-end HiSeq run.