title: GSM2478430: PBS2 WGBS; Mus musculus; Bisulfite-Seq
library_construction_protocol: P35 female mice were deeply anaesthetized with CO2 and then quickly perfused intracardially with ice-cold PBS. Whole brains were removed and stored on ice in HBSS w/out Ca/Mg until processed. Each brain was gently homogenized to a single cell solution using a dounce, then added to 1.8ml of isotonic Percoll to form a 30% isotonic Percoll solution.  Isotonic Percoll gradients were then constructed with layers of 70% with 1x phenol red, 37% and the top layer of brain/30%.  The resulting gradients were spun in a swinging bucket centrifuge for 30min at 500xg at room temperature. The top layer of myelin and non-microglial cells were discarded and the microglia at the interface between the 37% and 70% layers were collected. The resulting microglia were then washed in HBSS and counted.  An additional animal was run in parallel and harvested microglia were used to assess purity by flow cytometry for CD45.2 and CD11b. The collected cells were immediately flash frozen for RNA/DNA extraction. DNA was isolated from the microglia samples using ZR-Duet RNA/DNA kit (Zymo, D7001). WGBS libraries were constructed using the Illumina EpiGnome/TruSeq DNA Methylation kit (Illumina, EGMK81312) with indexing barcodes (Illumina, EGIDX81312). 100 ng of DNA from each sample was bisulfite converted using the EZ DNA Methylation-Lightning kit (Zymo, D5030.  Each library was given a unique barcode identifier and 14 cycles of PCR amplification. Libraries were quantified by Quibt and quality was assessed by Bioanalyzer. Sequencing of barcoded and pooled libraries was performed across two lanes of two runs on the Illumina HiSeq4000 to obtain 100 bp single end reads.
attributes.tag: GEO Accession
attributes.value: GSM2478430