title: GSM9086969: Primary human and mouse cerebellar NEPs neurosphere cultures; Homo sapiens; Mus musculus; RNA-Seq
library_construction_protocol: Primary NEP neurospheres were establised from postnatal day 1 old Nes-Cfp/+ or Swiss webster mouse cerebella, or fetal human cerebella obtained from HDRB and BRC (see details for the manuscript). Briefly, the this tissue was dissociated using Accutase (ThermoFisher, #A1110501) for 10-20 minutes at 37°C.Cells were filtered with a 40 µM nylon filter (Merck, #CLS352340) after dissociation to remove debris. Once a single cell suspension was obtained, cells were washed with NSC media (Neurobasal (ThermoFisher, #10888022) with 1X B27 (ThermoFisher, #A3353501), 1X N2 (ThermoFisher, #11520536), 2mM L-glutamine (ThermoFisher, #A2916801), 1X non-essential amino acids (ThermoFisher, 11140035) and 100 U/mL penicillin-streptomycin (ThermoFisher, #15140122)) and plated onto ultra-low attachment plates ~100,000 cells/mL density. Cultures were maintained with 20 ng/mL of EGF (Fisher Scientific, #PMG8041) and FGF2 (Qkine, #Qk042-0500) (or human EGF (QKINE, #Qk011-0100) and FGF2 (QKINE, #Qk027-0100) for human cultures). Growth factors were supplemented every other day and media was changed weekly.  For scRNA-seq analysis, the primary neurosphere cultres (passages <20) were dissociated  with Accutase and washed once in NSC media. The cell suspensions were filtered (Corning, #352235) and pelleted at 500g for 5 minutes at room temperature followed by a washed with 1x PBS + 0.04% bovine serum albumin. The single cell RNA-sequencing was performed on Chromium X/iX (10X Genomics) with the Next-GEM 3′ v3.1 abd CellPlex multiplexing (10X Genomics). A final amount of ~2000cells/sample were aimed when loading based on manufacturer's instructions. Below is a list of the samples and the CellPlex Oligos used for multiplexing.  Sample  Age     ID BRC_2584        9w      CMO301 BRC_2583        9w 4d   CMO302 BRC_2591        10w 5d  CMO303 BRC_2593        12w     CMO304 HDBR_18265      12w     CMO305 HDBR_18294      12w     CMO306 HDBR_18267      13w     CMO307 HDBR_18285      13w     CMO308 HDBR_18290      15w     CMO309 HDBR_18275      17w     CMO3010 SW   P1      CMO311 Nes-Cfp      P1      CMO312 cDNA amplification and library preparation was performed according to manufacturer's guidelines (10X Genomics Next-GEM 3' 3.1v) by Cambridge Stem Cell Institute NGS core. 28 base paired-end sequencing was performed on a NovaSeq X at Cancer Research UK Cambridge Institute.