GSM3543743: X13_Cx3Cr1-GFPonly; Mus musculus; RNA-Seq
Cortical microglia were isolated from four independent ∼12-week-old two- color animals (Cx3Cr1GFP/+; Hoxb8IRES-Cre/+; ROSA26CAG-LSL-tdTomato/+).  Animals were humanely euthanized by isoflurane, cortical tissue dissected and meninges removed, minced and trypsin digested (1% trypsin, 37°C, 30min in HBSS). Cortical single-cell suspensions were generated by mechanical trituration and successive filtration (70 and 30 μm mesh) and glia enriched via capture with anti-Cd11b (Itgam) magnetic beads (Miltenyi Biotec). Bead-associated cortical microglia were then sorted on a BD FACSAriaII gating on tdTomato and GFP with collection into RNAlater solution, flash frozen and stored at −80°C Total RNA was isolated and cloned by the University of Utah High-throughput Genomics Core using a Clontech UltraLow RNA isolation kit, and barcoded libraries sequenced on an Illumina HiSeq platform (50 bp nonstranded single-end reads), resulting in ∼25-30  million sequences per biological replicate. Sequence quality was confirmed by FastQC. Raw fastq reads were mapped using STAR (2.4.1c) (Dobin et al., 2013). Indices for STAR were built based upon the Gencode GRCm38v3 release M5 of the mouse genome. The index for STAR was constructed based upon the primary assembly and included splice junction information from the GTF file.
GEO Accession
GSM3543743