GSM8880879: LUAD,T1N0M0,sample1; Homo sapiens; RNA-Seq
The obtained tissue was transferred to a culture dish pre-added with 1×PBS (free of RNase and Ca, Mg ions) (the culture dish was placed on wet ice), and the tissue was cut into 0.5mm² small pieces. Then, the pieces were repeatedly washed with 1×PBS to remove surface blood stains, grease and other adherents. The washed small pieces of tissue were added with dissociation solution (0.35% collagenase IV, 2 mg/ml papain, 120 Units/ml DNase I), and reacted at 37°C on a water bath shaker (100 rpm) for 20 minutes. Then, 1×PBS (containing 10% fetal bovine serum FBS) was added to terminate the dissociation, and the cells were gently pipetted 5-10 times (with gentle operation to prevent cell death caused by shear force). The cell suspension was filtered through a 70-30um cell sieve, and then centrifuged at 300g for 5 minutes at 4°C. After centrifugation, the cell precipitate was collected and resuspended in 100ul 1×PBS (0.04% BSA) solution. To remove red blood cells, 1ml of red blood cell lysis buffer (MACS 130-094-183, 10×) was added, and the lysis was carried out at room temperature or on wet ice for 2-10 minutes. After lysis, the cells were centrifuged at 300g for 5 minutes at room temperature, and the cell precipitate was collected. Then, 50μl of dead cell removal reagent: Dead Cell Removal MicroBeads (MACS 130-090-101) was added and dead cells were removed using Miltenyi® Dead Cell Removal Kit (MACS 130-090-101). After the reaction, the reagent was removed by centrifugation. Subsequently, the cells were resuspended in 1×PBS (0.04% BSA) and centrifuged at 300g for 3 minutes at 4°C (repeated twice). The candidate cells after tissue dissociation, red blood cell lysis and dead cell removal were resuspended in 50μl 1×PBS (0.04% BSA) to form a cell suspension. Cell viability was detected by trypan blue staining, and the cell viability requirement was >85%. The cell count was determined by hemocytometer or cell automatic counter (Countess II Automated Cell Counter), and the cell concentration requirement was 700-1200 cells/μl. Add the prepared cell suspension and reaction mixture (Master Mix + Sample), 10× barcode gel beads (VDJ 3' Gel Beads v1.1), and partitioning oil to different wells of the Chromium Next GEM Chip, respectively. A GEM is formed via the microfluidic "single cross" system. Then, dissolve the gel beads and lyse the cells within each GEM. The released oligonucleotides are mixed with the lysed cells and Master Mix (containing RT reagents and RT primers) for reverse transcription to obtain full-length complementary DNA (cDNA) sequences with 10× barcodes. After reverse transcription, destroy the water-in-oil structure using a recovery agent, and purify the cDNA. Subsequently, amplify the cDNA using DNA polymerase. Then fragment the amplified cDNA and select appropriate fragments. After end repair and A-tailing, ligate Read2 sequencing primers, perform PCR, and construct a cDNA library with P5 and P7 adapters.