title: GSM8943908: E17.5 Emx1-Upf2cKO cortex; Mus musculus; RNA-Seq
library_construction_protocol: Single nuclei were isolated from E17.5 Emx1-Upf2cKO cortex and littermate control according to the 10x Genomics protocol (CG000124). In brief, the cortex tissue was minced on ice in 3 ml chilled lysis medium containing 0.05% Nonidet P40 Substitute for 5 minutes. Then 3 ml chilled HEB medium was added to the lysed cells and mixed 20 times with the fire-polished pipette. Next, the cell debris and clumps were removed by 30 μm MACS SmartStrainer. The nuclei pellet was collected after centrifugation at 500 rcf for 5 min at 4 ºC. Then the isolated nuclei were washed twice with 3 ml Nuclei Wash and Resuspension Buffer (1x PBS with 1% BSA and 0.2 U/μl RNase inhibitor). We resuspended with Nuclei Wash and Resuspension Buffer to get a concentration of 800 nuclei per μl in wild-type and 1200 nuclei per μl in Emx1-Upf2cKO. The single nuclei solutions were loaded into the single-cell chip with a target recovery of 10k cells in the 10x Genomic Chromium machine. After completing the 10x Genomics program, 100 µL of Gel Bead-in-Emulsions (GEMs) per sample were recovered for GEM-RT incubation. The resulting cDNA was purified, amplified over 12 PCR cycles, and subjected to QC, where samples passed if their fragment sizes fell within the 300–9,000 bp range. For library preparation, 20 µL of cDNA per sample was fragmented, followed by end repair and A-tailing. Adapters were then ligated, and samples underwent 14 additional PCR cycles using unique indices (either from the 10x index plate or custom IDT-synthesized indices). Post-amplification, PCR products were purified and size-selected (300–600 bp), with successful QC confirmation required for sequencing. Finally, libraries of equal mass were pooled for multiplexed sequencing.