title: GSM8901686: Synovium, hip OA, OA009; Homo sapiens; RNA-Seq
library_construction_protocol: Human FAI and hip OA synovium samples (3 samples per disease condition per sex) were harvested for scRNA-seq analysis. The FAI synovium samples were minced by hand and digested in 0.3% collagenase medium (DMEM/F-12, GlutaMAX™ supplement, #10-565-018, Gibco™ with 3 ug/mL collagenase II, #LS004177, Worthington Biochemical Corporation, and 10% Fetal Bovine Serum, #F0926-100, Sigma-Aldrich) for three hours at 37C while shaking continuously. The samples were thoroughly vortexed every hour. The OA synovium samples were digested in 0.4% collagenase medium (DMEM/F-12, GlutaMAX™ supplement, #10-565-018, Gibco™ with 4 ug/mL collagenase II, #LS004177, Worthington Biochemical Corporation, and 10% Fetal Bovine Serum, #F0926-100, Sigma-Aldrich) at 37C overnight. Cells were then resuspended in ACK lysing buffer (#A1049201, Gibco) for 1-2 minutes to lyse red blood cells. Afterwards, cells were filtered through 70um (#130-110-916, Miltenyi Biotec) and 30um MACS® SmartStrainers (#130-110-915, Miltenyi Biotec). The cell suspension was later frozen down in freezing medium (DMEM/F-12, GlutaMAX™ supplement, #10-565-018, Gibco™ with 10% Pierce™ Dimethylsulfoxide (DMSO), LC-MS Grade, #85190, ThermoFischer, and 80% Fetal Bovine Serum, #F0926-100, Sigma-Aldrich) until submitted for scRNA-seq. Samples with greater than 70% viability were submitted to UR Genomics Research Center for scRNA-Seq library preparation using Chromium Next GEM Single Cell 3′ GEM, Library and Gel Bead Kit v3.1 (10x Genomics), per the manufacturer's recommendations, as summarized below. Samples were loaded on a Chromium Single-Cell Instrument (10x Genomics, Pleasanton, CA, USA) to generate single-cell GEMs (Gel Bead-in-Emulsions). GEM reverse transcription (GEM-RT) was performed to produce a barcoded, full-length cDNA from poly-adenylated mRNA. After incubation, GEMs were broken, the pooled GEM-RT reaction mixtures were recovered, and cDNA was purified with silane magnetic beads (DynaBeads MyOne Silane Beads, ThermoFisher Scientific). The purified cDNA was further amplified by PCR to generate sufficient material for library construction. Enzymatic fragmentation and size selection were used to optimize the cDNA amplicon size and indexed sequencing libraries were constructed by end repair, A-tailing, adaptor ligation, and PCR. The final libraries contain the P5 and P7 priming sites used in Illumina bridge amplification. Sequence data was generated using the Illumina NovaSeq 6000 platform.
identifiers.namespace: GEO
identifiers.id: GSM8901686_r1