GSM8994143: sample 5 laryngeal squamous cell; Homo sapiens; RNA-Seq
Samples were chopped into 1 mm³ pieces using a sterile scalpel and then mixed in 5 ml of a digestion buffer that included 2 mg/mL Trypsin (ThermoFisher, 15400054), 2 mg/mL Collagenase type II (Sigma, C6885), and 200 U/ml DNase I (Worthington, LS006344) in RPMI medium (Coring, 10-040-CV). This mixture was incubated in a 37℃ water bath with shaking for 30 minutes. The resulting suspension was filtered through a 100 μm filter (Falcon, 3523260) and centrifuged at 400g for 10 minutes at 4℃. The pellet was then resuspended in red blood cell lysis buffer (Solarbio, R1010) and incubated for 2 minutes before being filtered through a 40 μm filter (Falcon, 3523240). After another centrifugation (400g, 10 minutes, 4℃), the cells were resuspended in PBS (BI, 02-024-1ACS) containing 0.04% BSA (Sigma, B2064). Cell counts were performed manually using Trypan blue (Thermo, T10282) and AO-PI (LUNA, D23001) after each centrifugation and resuspension. Single cells were then processed with the Chromium Controller (10X Genomics) following the manufacturer's instructions. We conducted single-cell 3' gene expression profiling using the Chromium Next GEM Single Cell 3ʹ Kit v3.1 (10x Genomics, 1000268) and the Chromium Next GEM Chip G Single Cell Kit (10x Genomics, 1000120). The cell suspension was placed into the Chromium single cell controller (10x Genomics) to create single-cell gel beads in emulsion, following the manufacturer's instructions. The captured cells were lysed, and the released RNA was barcoded through reverse transcription within individual GEMs. The cell-barcoded 3' gene expression libraries were sequenced on an Illumina NovaSeq6000 system (Illumina, San Diego, CA, USA) by Shanghai Biochip Co., Ltd., located in Shanghai, China.