GSM8942351: ex_vivo_expanded_cKITenriched_cells_mouse3_L2_HTO; Mus musculus; OTHER
For CITE-seq, cell suspensions containing 2.5 × 10⁶ cells per sample were pooled and centrifuged at 300 × g at 4°C for 10 minutes. Each pool included a sample from each tested condition, with replicates separated across different pools to control for batch effects. Next, cells were resuspended in 100 µL PBS and filtered through 40 µm Flowmi™ cell strainers (Scienceware). Cell density and viability were assessed using an EVE™ Automatic Cell Counter (NanoEnTek Inc., USA). If necessary, the cell suspension was diluted to a final density of 2,000 – 10,000 cells/µL. Sample pools were processed in quadruplicates, with each replicate loaded onto a separate chip well (Chip G, 10x Genomics). Cell encapsulation and library preparation were performed using the Chromium Controller (10x Genomics) and the Chromium Next GEM Single Cell 3' Reagent Kit v3.1 with Feature Barcoding technology for Cell Surface Protein (10x Genomics), following the manufacturer's user guide (CG000206 Rev D). Library quality and quantity were assessed using a High-Sensitivity DNA Kit (Agilent Technologies, USA) on a 2100 Bioanalyzer (Agilent Technologies, USA). Samples were sequenced on a NovaSeq 6000 in paired-end mode (Read 1: 28 cycles, i7 index read: 8 cycles, i5 index read: 0 cycles, Read 2: 91 cycles). The sequencing depth was targeted at 4 × 10⁴ mRNA reads and 7 × 10³ Ab-oligo reads per cell.