GSM9289190: Gestational weeks 13 telencephalon 2; Homo sapiens; RNA-Seq
tissue samples were dissociated using the Papain Dissociation System (Worthington, LK003150), dissociated cells were centrifuged, the supernatant removed, and the pellet resuspended in ice-cold PBS supplemented with 0.04% BSA. After a second centrifugation and supernatant removal, the cell pellet was resuspended in 1 mL of Fix & Perm Buffer (room temperature) and incubated for 17.5 h at 4 °C without agitation. Cells were then centrifuged at 850 × g for 5 min at room temperature, the supernatant removed, and 1 mL of chilled Quenching Buffer added and mixed by pipetting (5×). Next, 100 μL of pre-warmed Enhancer was added, followed by glycerol to a final concentration of 10%. Cells were subsequently fixed with the Chromium Next GEM Single Cell Fixed RNA Sample Preparation Kit (10x Genomics). Single-cell gene expression libraries were prepared with the 10x Genomics Chromium Single Cell Gene Expression Flex Kit (PN-1000414) following the manufacturer's protocol (CG000527 Rev E). Approximately 40,000 cells per sample were loaded onto a Chromium Next GEM Chip and processed on the Chromium Controller to generate Gel Bead-in-Emulsions (GEMs) for probe-based capture, reverse transcription, and barcoding of mRNA with unique molecular identifiers (UMIs) and cell barcodes. After cDNA amplification, libraries were constructed by fragmentation, end repair, A-tailing, adapter ligation, and final PCR enrichment. Library quality and concentration were assessed on an Agilent 2100 Bioanalyzer and a Thermo Fisher Qubit 4 Fluorometer. Pooled libraries were sequenced on an Illumina NovaSeq X, targeting ~20,000 reads per cell.