title: GSM6567166: ADJ2, scRNAseq; Homo sapiens; RNA-Seq
library_construction_protocol: All fresh resection specimens were preserved in the tissue storage solution (Miltenyi, Cat. no. 130-100-008) on ice and transported to the laboratory in CapitalBio Technology company within 1.5-2 hours. To ensure the success of tissue dissociation, multiple resection specimens from the same patients were digested. All specimens were cut into around 1 mm pieces and incubated in an optimal digestive solution, including enzyme cocktail, consisting of the Type VIII Collagenase (Sigma-Aldrich, Cat. no. C2139), DNase I (Sigma-Aldrich, Cat. no. D5025), trypsin inhibitor (Sigma-Aldrich, Cat. no. T6522), and Dispase II (neutral protease, grade II, Sigma-Aldrich, Cat. no. 4942078001). The single cell suspension was filtered with a 40 μm cell strainer (BD, Cat. no. 352340), then incubated with red blood cell lysis buffer (Roche, Cat. no. 11814389001) at 4°C for 10 minutes. The concentration of single cell suspension was determined using the Count Star instrument and adjusted to 1000 cells/μl. The cell suspension was loaded onto the Chromium single cell controller (10x Genomics) to generate single-cell gel beads in the emulsion using single cell 3' Library and Gel Bead Kit v3.1 (10x Genomics, Cat. No. 1000121) and Chromium Next GEM Chip G Single Cell Kit (10x Genomics, Cat. No. 1000120) according to the manufacturer's instructions. Briefly, cells were suspended in PBS containing 0.04% BSA. About 6,000 cells were loaded to each channel, and the target cell will be captured about 3000 cells per channel. Captured cells were lysed and the released RNA were barcoded by reverse transcription in each GEM. Reverse transcription was completed in 200 ul tubes (NEST Biotechnology, Cat. No. 401001) on a S1000TM Touch Thermal Cycler (Bio Rad) at 53°C for 45 minutes, then at 85°C for 5 min, and hold at 4°C. The cDNA libraries were generated, amplified, and quality assessed using the Agilent 4200. Finally, the cDNA libraries were sequenced using an Illumina Novaseq6000 sequencer with a sequencing depth of at least 100,000 reads per cell with paired-end 150 bp (PE150) reading strategy.