title: GSM8600001: Synovium, Immunotype 2, Sample 93, scRNA-seq; Homo sapiens; RNA-Seq
library_construction_protocol: At the time of ACL reconstruction, an arthroscopic synovial biopsy (200-300 mg) from the prefemoral synovium was obtained. Synovial tissue was enzymatically digested with an RPMI 1640 (Cytiva SH30027.02) media solution containing 4 mg/mL collagenase type 4 (Worthington Biochemical LS004189), 0.8 mg/mL dispase II (Millipore Sigma 04942078001), plus 0.1 mg/mL DNase I (Millipore Sigma 11284932001). Digest was passed through a 70 µm filter to obtain a single cell suspension. For cell suspensions containing >20% erythrocytes, lysis was performed with RBC lysis buffer (BD 555899) according to the manufacturer's instructions. 60,000 cells were set aside for scRNA-seq library preparation. Single cell partitioning and RNA library preparation were performed by the the Atrium Health Molecular Biology Core Facility  on the same day as cell isolation (separate days for each of the six samples). Nanoliter-scale Gel Beads-in-emulsion (GEMs) were generated consisting of barcoded single cell 3ʹ v3.1 gel beads, a master mix containing cells, and partitioning oil onto Chromium next GEM chip G (10x Genomics). Thereafter, the gel bead was dissolved resulting in the release of the primers. cDNA was produced using the poly(dT) primer included on the gel beads. Each cell's transcriptome was indexed using a pool of individual 10x barcodes whereas all generated cDNA shared a common 10x barcode. Barcoded, full-length cDNA was then PCR amplified, enzymatically fragmented, and size selected to the optimum amplicon length. Following end repair, A-tailing, adaptor ligation, and PCR, the final libraries contained Illumina adaptors (P5, P7), primers (Read1, Read2), and index sequences which were sequenced with 26 cycles for read1, and 98 cycles for read2. Sequencing was performed in two batches on separate days. Batch 1 included Sample 78, Sample 80, and Sample 84. Batch 2 included Sample 85, Sample 93, and Sample 95.