GSM9151151: Patient 1, tumor tissue; Homo sapiens; RNA-Seq
First, the samples were washed three times with PBS. The tissues were completely minced with scissors, and tissue digestion solution was added, followed by digestion on a shaker at 37ºC for 60 minutes. The resulting cell suspension was filtered through 100μm and 40μm strainers. After low-speed centrifugation to pellet the cells, red blood cell lysis buffer was used to remove red blood cells. Subsequently, the cell concentration was adjusted to the optimal level for the subsequent library construction steps. Single-cell RNA-Seq libraries were prepared using the SeekOne® DD kit. Cells were mixed with reverse transcription reagent and added to Chip S3. Barcoded Hydrogel Beads and partitioning oil were dispensed into Chip S3 wells. Reverse transcription was performed at 42°C for 90 min and inactivated at 85°C for 5 min. cDNA was purified, amplified in polymerase chain reaction (PCR), cleaned, fragmented, repaired, A-tailed, and ligated to a sequencing adaptor. Indexed PCR was then performed to amplify the DNA representing the 3' polyA part of expressing genes, containing cell barcode and unique molecular index. Libraries were cleaned with solid phase reversible immobilization beads, quantified by qPCR, and sequenced on DNBSEQ-T7 with PE150 read length.