title: GSM3211706: Amplicon_BS_Serum_90; Mus musculus; Bisulfite-Seq
library_construction_protocol: Cells were lysed by removing media from culture dishes and adding 200ul of RLT plus buffer (Qiagen) supplemented with 0.5mM 2-mercaptoethanol. . Total nucleic acid was purified from cell lysates with RNAdvance magnetic beads (Beckman Coulter, A32649) using a Bravo Workstation pipetting robot (Agilent Technologies) following the manufacturers protocol. RNA was subsequently purified by treating the total nucleic acid with DNase I. KAPA HiFi Uracil+ Master Mix (Kapa Biosystems) was used to amplify regions of interest using 30nM primers (Table S3), with the reverse primer including an 8N unique molecular identifier (UMI). 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 (Table S5) and iPCRTag primers (Quail et al., 2012), 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 MiSeq run.
attributes.tag: GEO Accession
attributes.value: GSM3211706