GSM7153012: C5_E14.5 Control; Mus musculus; RNA-Seq
Cortex of E12.5 and E14.5 embryos was dissected and dissociated into single cells using the Papain Dissociation System (#LK0031500, Worthington Biochemical). Individual collected cells were processed for scRNAseq using. Library was performed according to the manufacter's instructions (single cell 3' v3.1 protocol, 10x Genomics). Briefly, The NextGEM protocol was run on the 10X Chromium Controller to create GEMs, composed of a single cell, gel bead with unique barcode and UMI primer, and RT reagents. 100 µl of emulsion is retrieved from the chip and incubated (45 min at 53°C, 5 min at 85°C, cool to 4°C), generating barcoded cDNA from each cell. The GEMs were broken using Recovery Agent and cDNA cleaned, following manufacturer's instructions using MyOne SILANE beads. cDNA was amplified for 11 cycles (3 min at 98°C, 11 cycle: 15 sec at 98°C, 20 sec at 63°C, 1 min at 72°C; 1 min at 72°C, cool to 4°C).  Samples were cleaned using 0.6X SPRIselect beads. Quality control (QC) was completed using Qubit and Bioanalyzer to determine size and concentrations. 10 µl of amplified cDNA was carried into library prep. Fragmentation, end repair and A-tailing were completed (5 min at 32°C, 30 min at 65°C, cool to 4°C), and samples were cleaned up using double sided size selection (0.6X, 0.8X) with SPRIselect beads. Adaptor ligation (15 min at 20°C, cool to 4°C), 0.8X cleanup and amplification were performed by PCR using unique i7 index sequences. Libraries underwent a final cleanup using double sided size selection (0.6X, 0.8X) with SPRIselect beads. Library QC was performed using Qubit, Bioanalyzer and KAPA library quantification qPCR kit.