GSM2477583: MAA2 RNAseq; Mus musculus; RNA-Seq
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. Duet RNA/DNA kit (Zymo, D7001) with on-column DNase digestion (Qiagen, 79254) for RNA extraction.  RNA and DNA were quantified using Qubit High Sensitivity assays.  RNA quality was assessed by Bioanalyzer and only samples with RIN scores greater than 7 were used for library preparation.  60-100 ng of input RNA per sample were used for RiboGone rRNA removal (Clonetech, 634847).  2 ng of rRNA depleted RNA per sample was then used for library construction using the SMARTer Stranded RNA-Seq kit (Clontech, 634836) and 14 cycles of PCR amplification.  Library quality was assessed using a bioanalyzer and quantified by Qubit high sensitivity assay. All samples were barcoded, pooled, and run on a single sequencing lane of a HiSeq3000 to generate 50 bp single end reads.
GEO Accession
GSM2477583