GSM8124170: T4, replicate2, scRNAseq; Homo sapiens; RNA-Seq
Lung tissue samples were washed with phosphate buffer solution (PBS) and immersed in DMEM (Gibco, #11885084, USA) supplemented with 10% fetal bovine serum (Gibco, #10091148, USA). Samples were cut into small pieces. After washing with PBS, samples were digested in PBS containing 1000 U/mL Collagenase II (Worthington, #43J14367B, USA) at 37°C for 15 minutes with gentle shaking. The above steps were repeated 3 times. The cell suspension was filtered with a 40 μm cell strainer (Falcon, #431750, USA) to get a single-cell suspension. Cells were collected by centrifugation at 400 g, 4°C for 5 minutes. The supernatant was discarded. The cell pellets were re-suspended in DMEM containing 2% fetal bovine serum. The cell pellet was treated with 200 μL red blood cell lysis buffer (Beyotime, #C3702, China) for 10 minutes on ice. After centrifugation, the suspension was re-suspended. Single-cell suspension was harvested again with a 40 μm cell strainer. Single-cell suspensions were loaded onto a Chromium Single Cell Controller (10x Genomics) to generate Gel Bead-In-Emulsions (GEMs). cDNA libraries were prepared using the single cell 5' solution v2 reagent kit (Chromium, 1000020) according to the protocol provided in the 10x Genomics Chromium Single Cell Immune Profiling Solution. After the reverse transcription step, droplets were disrupted and barcoded cDNAs were purified with DynaBeads, followed by 14 cycles of PCR amplification (98°C for 45 seconds; [98°C for 20 seconds, 67°C for 30 seconds, and 72°C for 1 minute]×14 cycles; 72°C for 1 minute). The resulting amplified cDNAs were sufficient to construct 5′ gene expression libraries. The cDNAs from single-cell transcriptomes (50 ng) were fragmented, subjected to 2 rounds of size selection with SPRI beads (avg. size 450 bp), and sequenced on the Illumina NextSeq platform (High Output V2 Kit, 150 cycles). All libraries were sequenced by an Illumina HiSeq 4000 sequencer.