scRNA-Seq of Homo sapiens:ESCC_met3
The tissue samples were rinsed with phosphate-buffered saline (PBS), minced into small pieces on ice, and the tumor tissue was further fragmented and incubated with 0.5 mg/mL of Digestion Enzyme 1 (prepared by 10K Genomics, Shanghai, China) at 37C in a water bath for 60 min. The suspension was loaded onto a 10 Chromium Controller (10 Genomics) following the manufacturers instructions. Single-cell RNA-seq libraries were prepared using the Chromium Single-Cell 3-End V3.1 Kit (Catalog No.: CG000204 Rev D). Single-cell suspensions were processed using a 10 Chromium Controller to generate emulsion droplets, where individual cells in each droplet interacted with gel beads containing unique primers bearing 10 cell barcodes, unique molecular identifiers, and poly(dT) sequences. Full-length barcoded cDNA was synthesized using reverse transcription. Subsequently, the emulsion was disrupted, and cDNA was harvested, amplified, and enriched using reagents from the Chromium Single-Cell 3 V3.1 Kit. Indexed sequencing libraries were constructed as follows: (1) fragmentation, end repair, and A-tailing; (2) size selection of cDNA fragments using SPRI beads; (3) ligation of sequencing adapters; (4) further enrichment of cDNA fragments with SPRI beads; and (5) indexing and final library enrichment using SPRI beads. The number of libraries sequenced was determined based on the cell count and paired-end 150-bp sequencing was performed on NovaSeq 6000 (Illumina).
SUB15663191
P11_1