GSM3904824: brain cortex, Juvenile replicate2; Mus musculus; RNA-Seq
Mice were perfused transcardially with ice-cold PBS under anesthesia. Brain cortex tissues were dissected and digested using the enzyme-cocktail solution of 30U/ml papain (LK003153; Worthington) and 0.125mg/ml Liberase TM (5401119001; Sigma-Aldrich) in DMEM (Sigma-Aldrich) for 60 min at 37 °C. Homogenized tissues were added by 1.7-fold volume of 22% bovine serum albumin in PBS and then centrifuged at 1,000 rcf for 10 min for myelin removal. After washing with ice-cold DMEM, dead cells and debris were removed using ClioCell nanoparticles (amsbio) according to the manufacturer's instructions. Single cells were counted using Luna-II automated cell counter (Logos Biosystems) and captured using the 10X Chromium system (10X Genomics). We pooled tissues from three (for Aged and EAE samples) or six (for Juvenile and Adult samples) mice to reduce individual variations. Libraries were prepared according to the manufacturer's instructions using Chromium Single Cell 3' Library & Gel Bead Kit v2 (10X Genomics) 3' single cell RNA-Seq using cell barcodes and unique molecular identifiers (UMIs).