Name: GSM6779221
Instrument: Illumina HiSeq 2500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC SINGLE CELL
Selection: cDNA
Layout: PAIRED
Construction protocol: After microdissecting individual germinal zones from living cortical slices, tissue pieces were enzymatically dissociated for 20-30' at 37ºC using MACS Neural Tissue Dissociation Kit P (Miltenyi Biotec) according to the manufacturer's instructions. Cells were mechanically suspended using wide-tipped, fire-polished Pasteur pipettes (Fisher Scientific) followed by P1000 pipettes (Gilson). Cell suspensions were filtered to 2ml DNA LoBind tubes (Eppendorf) through 40 μm PluriStrainer Mini (PluriSelect) pre-wet with Hank's Balanced Salt Solution (1X) without CaCl2/MgCl2/pH indicator (HBSS, Gibco). Cells were centrifuged (Spectrafuge 24D, Labnet) twice for 5min at 1000 rpm, and cell pellets were resuspended in 40μl of Phosphate Buffered Saline ph7.4 (1X) without CaCl2/MgCl2 (PBS, Gibco) containing 0.04% Bovine Serum Albumin (BSA, Sigma-Aldrich). Cell concentration from homogeneous suspensions was measured, and cell death was estimated twice using trypan blue 0.4% stain (Thermo Fisher Scientific): first on an automatic cell counter (Countess II FL, Thermo Fisher Scientific) and second using a hemocytometer (BLAUBRAND Neubauer, BRAND) on an inverted microscope (Leica DM IL). Debris-free suspensions with cell viability over 90% were used immediately for single-cell isolation. Cell suspensions from 400 to 1200 cells/μl were loaded into the Chromium Single Cell Controller (10x Genomics) to target a recovery of 7k to 10k cells/sample. Single-cell Gel bead-in-EMulsions (GEMs) were generated, cells were lysed, and the released RNAs were retrotranscribed (RT) and barcoded inside the GEMs according to Single Cell 3' Reagent Kits v2 and v3 (10x Genomics) protocols. Following GEMs break, barcoded cDNAs were pooled and cleaned from leftover RT reagents using DynaBeads MyOne Silane Beads (Invitrogen) and amplified (8 to 14 cDNA amplification cycles) following the Single Cell 3' Reagent Kits v2 and v3 (10x Genomics) protocols. Enzymatic fragmentation, size selection and cleanup with SPRIselect Reagent kit (Beckman Coulter) were followed by library construction (total sample index cycles from 12 to 16 cycles), consistent with standard Illumina sequencing constructs and performed in line with Single Cell 3' Reagent Kits v2 and v3 (10x Genomics) protocols. cDNA and library concentration and quality check were assessed using Bioanalyzer High Sensitivity DNA Kit (Agilent) and 2100 Expert Software (Agilent).