GSM9157700: late _stim; Homo sapiens; RNA-Seq
To collect cells, plates were washed once in cold PBS, cells were scraped in cold PBS and then centrifuged for 3 minutes at 250 xg. Cell pellets were resuspended in Synthafreeze (Gibco, A1254201), transferred to a cryotube, and slow-froze to -80 ºC. Samples were recovered by thawing at room temperature and extracting nuclei via sucrose gradient centrifugation Upon nuclei isolation, nuclei were resuspended in FANS Buffer (0.2% BSA in PBS, supplemented with protease inhibitor and RNAse inhibitor (Invitrogen, AM2696)), stained with 1:1000 DAPI (Sigma-Aldrich, D9542-1MG) and sorted on an AurorsaCS-2 (Cytek) sorter with a 70 µm nozzle. High-quality nuclei populations were identified by sorting first on forward-scatter area and side scatter area to remove debris, then forward-scatter area and forward-scatter heigh to identify single nuclei, then side-scatter area and UV7 area to identify DAPI positive nuclei using SpectroFlo CS (Cytek) software. High quality nuclei fell into a low variability positive sub population within the UV7 area measurement. After sorting, nuclei were counted and diluted to 700 nuclei/µL in FANS buffer. To generate droplets containing a single nuclei per droplet, the Chromium Next GEM Chip G Single Cell Kit (10X Genomics, PN-1000120) on the Chromium Controller (10X Genomics, PN-120270) was used, targeting the recovery of 10,000 nuclei per condition. Single nucleus RNA seq was then performed using the Chromium Next GEM Single Cell 3' Reagent Kit v3.1 (10X Genomics, PN-1000268) following the manufacturers protocol. Samples were indexed using the Dual Index Kit TT Set A (10X Genomics, PN-1000127), and libraries were amplified using 15 PCR cycles. Samples were sequenced on the NextSeq 1000 P2 flow cell (100 cycles; Illumina, 20046811), setting Read 1 to 28 cycles and Read 2 to 90 cycles.