title: GSM7817744: Het 3 scRNAseq; Mus musculus; RNA-Seq
library_construction_protocol: Timed-pregnant females were sacrificed to collect embryos (E16.5). Brains were collected from the embryos (n=3 for WT and KO, n=4 for Het), then the cortex was carefully removed using a stereo microscope from each embryo and placed in a 15 ml tube containing Hibernate E/B27/GlutaMax (HEB) media (BrainBits). Tissue pieces from the cortex were then incubated in a Papain solution (BrainBits) at 37°C for 20 minutes. Following this, the Papain solution was gently removed and replaced with the HEB media. The tissues were manually disrupted using a polished Pasteur pipette for a maximum of 10 times. After disruption we allowed the tissue debris to settle for 1 minute at room temperature, the supernatant was transferred to a new tube and centrifuged at 200 rcf for 2 minutes to collect the cells. The supernatant was then carefully removed, and the cell pellet was resuspended in 1 ml of pre-warmed neuronal culture medium (NbActiv1). Subsequently the cell suspension was passed through a 30 µM cell strainer (Miltenyi Biotech) and an aliquot was used for counting using the Automated cell counter (Invitrogen) and morphological evaluation. Single cell suspensions containing more than 90% viable cells were used for the generation of single-cell RNA sequencing libraries. Fast genotyping (~1h) was performed with tissue from the rest of the body using Platinum Direct PCR Universal Master Mix Single-cell RNA-seq libraries were generated using the 10X Genomics platform (Chromium Single Cell 3′ library and Gel Bead Kit v3, PN-1000075) and sequenced on a NovaSeq 6000 system (Illumina) at the Center for Genome Technology at the HIHG. Cells at a concentration of 1200 cell/mL were loaded on the 10X Genomics Chromium platform to isolate ~7,000 cells per sample and create individually barcoded Gel bead-in-Emulsions (GEMs) which were processed using the Chromium Single Cell 3' Reagent Version 3 Kit. Sequencing libraries were evaluated for quality on the Agilent Tape Station (Agilent Technologies, Palo Alto, CA, USA), and quantified using a Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) and qPCR before sequencing on the Illumina NovaSeq 6000 targeting 100,000 reads per cell with sequencing parameters: Read1, 28 cycles; Index1, 8 cycles; Read2, 98 cycles.