GSM3466727: FB_RNA_rep2; Mus musculus; RNA-Seq
Pooled brain regions were dissociated using the Papain Dissociation System (Worthington Biochemical Corporation). The tissue was dissociated in the papain solution for 30 minutes at 37°C, with gentle trituration every 10 minutes using a sterile Pasteur pipette. Following dissociation, cells were passed through a 40µm cell strainer into a 50mL conical, centrifuged for 5 minutes at 300g, resuspended in albumin-inhibitor solution containing DNase, applied to a discontinuous density gradient, and centrifuged for 6 minutes at 70g. The resulting cell pellet was resuspended in HBSS with Mg2+ and Ca2+ and submitted to FACS. Aliquots of 50,000 EGFP+ cells were sorted directly into 300µL HBSS with Mg2+ and Ca2+ with 10% FBS for ATAC-seq. Aliquots containing ≥50,000 EGFP+ cells were sorted into kit-provided lysis buffer for RNA-seq. Total RNA was extracted using the Purelink RNA Micro Kit (Invitrogen). Following FACS isolation into kit-provided lysis buffer, samples were homogenized and RNA extraction proceeded using manufacturer's recommendations. Total RNA integrity was determined using the RNA Pico Kit (Agilent). RNA samples were sent to the Sidney Kimmel Comprehensive Cancer Center Next Generation Sequencing Core at Johns Hopkins for library preparation, using the Ovation RNA-Seq System V2 (Nugen), and sequencing.
GEO Accession
GSM3466727