GSM9973946: scRNA‑Seq + Cell Hashing;rep2;MCF-7; HTO; Homo sapiens; RNA-Seq
Cells were harvested using 0.05% Trypsin-EDTA (Gibco, Thermo Fisher Scientific Inc., USA), then washed twice with PBS and resuspended at a concentration of 2 x 10⁷ cells/ml in flow cytometry staining buffer. Human TruStain FcX (BioLegend, USA) was added at 5 μl per 1 million cells, and the samples were incubated at 4 °C for 10 minutes. Subsequently, 1 μg of TotalSeq™ anti-human Hashtag antibody (BioLegend, USA) (DMF: TotalSeq™-A0253 Hashtag 3 (TTCCGCCTCTCTTTG), niclosamide: TotalSeq™-A0254 Hashtag 4 (AGTAAGTTCAGCGTA)) was diluted in 50 μl of flow cytometry staining buffer, centrifuged at 14,000 x g at 4 °C for 10 minutes, and then added to the samples, which were incubated at 4 °C for 30 minutes. After incubation, 3.5 ml of flow cytometry staining buffer was added to each sample, and the samples were centrifuged at 250 x g at 4 °C for 5 minutes. This wash step was repeated twice. The cells were then resuspended in 400 μl of PBS containing 0.04% BSA (Sigma-Aldrich, USA), filtered through a 40 μm cell strainer (Corning Inc., USA), and counted using a TC10 Automated Cell Counter (Bio-Rad Laboratories Inc., USA). The cells were diluted to a final concentration of 1 x 10⁶ cells/ml, and equal volumes of the different samples were mixed. Cells underwent Chromium Single Cell 3' sample preparation following the manufacturer's instructions (v3, CG000185 Rev B, or v3.1, CG000206 Rev D) (10x Genomics, USA), together with hashtag oligo (HTO) cDNA amplification.