GSM7996336
The tissues were cut into small pieces in prechilled phosphate-buffered saline (PBS) and digested with 200 U/ml collagenase type II (Worthington, 43J14367B) in a 37°C water bath with mild shaking. The single-cell suspension was filtered with a 40 μm cell strainer and collected by centrifugation at 400 × g for 5 min. The supernatant was discarded, and the cell pellets were resuspended in 1 ml of 10% FBS/DMEM scRNA-Seq library preparation for 10x Genomics single-cell 5′ and suspensions were loaded on a Chromium Single-Cell Controller (10x Genomics) to generate a single-cell and gel bead emulsion (GEM).The scRΝΑ-seq libraries were prepared using a single-cell 5' solution v2 reagent kit (Chromium, 1000006) according to the protocol provided with the 10x Genomics Chromium Single-Cell Immune Profiling Solution.Briefly, FACS-sorted Cd45+ immune cells (90-95% viability) were encapsulated into droplets. Following the RT step, the droplets were disrupted and the barcoded cDNAs were purified with DynaBeads and subjected to 14 cycles of PCR amplification (98°C for 45 s; [98°C for 20 s, 67°C for 30 s, and 72°C for 1 min] x 14 cycles; 72°C for 1 min). The resulting amplified cDNAs were sufficient to construct 5' gene expression libraries and T/B cell receptor (TCR/BCR)-enriched libraries. The cDNAs from the single-cell transcriptomes (50 ng) were fragmented, subjected to two rounds of size selection with SPRI beads (average size 450 bp).