GSM8379692: Rep3, mRNA; Mus musculus; RNA-Seq
Mice were sacrificed and subsequently brains were collected, placed in cold HBSS (without Ca2+ and Mg2+), and transferred into 30 % sucrose (w/v) in PBS for 48 h and frozen in Tissue Freezing Medium (Leica) at -80°C. The tissue was minced, transferred onto c-tubes containing 1,950 µl DMEM, and supplemented with 50 µl of Deoxyribonuclease I (Sigma Aldrich). Dissociation was performed with the MACS dissociator with heaters (Militenyi Biotec) for 22 minutes and later the samples were passed through 70 nm and 40 nm strainers into 50 ml cold HBSS with ions to stop the enzymatic reaction (EasyStrainer™, BioOne). For flow cytometry, cells were suspended in the Stain Buffer (BD Pharmingen) with anti-mouse CD16/CD32 Fc Block™ 1:200 (BD Pharmigen) for 10 min. Next, anti-mouse CD11b antibody 1:250 (M1/70, BD Pharmigen) was added and cells were incubated for 20 min at 4 °C, then washed with Stain Buffer. Cells were sorted using purity precision mode on FACS Aria (BD FACSAria Cell sorter BD-Biosciences), into 200 μL of sterile PBS. Preparation of gel beads in emulsion andlibraries were performed with Chromium Controller and Single-Cell Gene Expression v2 Chemistry (10X Genomics). Libraries' quality and quantity were verified with a High-Sensitivity DNA Kit (Agilent Technologies, USA) on a 2100 Bioanalyzer (Agilent Technologies, USA).