GSM3402960: WT_clone_1_NPC (RRBS); Mus musculus; Bisulfite-Seq
Genomic DNA isolated with DNeasy Blood & Tissue Kit (QIAGEN) or Nucleon BACC2 Genomic DNA Extraction Kit (illustra) and quantified by Qubit (Invitrogen). DNA from mouse cortex samples were concentrated using Agencourt AMPure XP technology. 200ng of purified DNA samples were processed using the Ovation RRBS Methyl-Seq system kit according to instructions with the following modifications (NuGen Technologies). Briefly, unmethylated phage λ DNA 0.5ng (NEB) was spiked into each sample to allow assessment of bisulfite conversion efficiency. The methylation-insensitive restriction enzyme MspI was then used to digest the gDNA, and digested fragments were ligated to adapters. Adapter ligated fragments were then repaired before bisulfite conversion with the Qiagen Epitect fast bisulfite conversion kit. Bisulfite-treated adapter-ligated fragments were amplified by 9 cycles of PCR and purified using Agencourt RNAClean XP beads. Libraries were quantified using the Qubit dsDNA HS assay and assessed for size and quality using the Agilent Bioanalyzer DNA HS kit. Sequencing was performed using the NextSeq 500/550 high-output version 2 kit (75bp paired end reads) on the Illumina NextSeq 550 platform. As instructed for the NuGen RRBS kit, 12bp index reads were generated to sequence the Unique Molecular Identifiers (UMI) in addition to the index present in the adaptors. Libraries were combined into equimolar pools and run on a single flow cell. 10% PhiX control library was spiked in to facilitate sequencing by generating additional sequence diversity. Library preparation and sequencing performed by WTCRF, Edinburgh.
GEO Accession
GSM3402960