GSM8031359: synovial cells, Decitabine- KBxN, replicate 3; Mus musculus; RNA-Seq
For sc-RNA-seq, synovial cells were isolated from paw and ankle joints (n=3 per group). Briefly, at the end of the experiment, animals were sacrificed, and paw and ankle joints were dissected. Skin from the paw and ankle joints was removed, and the tissues were chopped in 50 ml tube containing digestion solution 2mg/ml Collagenase D solution, 2mg/ml Dispase and 1mg/ml DNase I. Tissues were digested for 30 min at 37oC in shaking water bath (250rpm). After 30 minutes, samples were vortexed briefly and supernatant was collected through a 70u strainer. This digestion cycle was repeated thrice. At the end of three times digestion, cells were centrifuged and suspended in appropriate buffer and processed for further analysis. 10X chromium platform scRNA-seq with cell hashing Three samples were collected from each experimental condition (i.e., control, STIA, STIA_DAC and DAC groups). cDNA was prepared after the GEM generation and barcoding, followed by the GEM-RT reaction and bead cleanup steps. Purified cDNA was amplified for 11-13 cycles before being cleaned up using SPRIselect beads. Samples were then run on a Bioanalyzer to determine the cDNA concentration. Gene Expression and Feature libraries were prepared as recommended by the 10x Genomics Chromium Single Cell 3' Reagent Kits User Guide (v3.1 Chemistry Dual Index) with Feature Barcoding Technology for Cell Surface Protein with appropriate modifications to the PCR cycles based on the calculated cDNA concentration. For sample preparation on the 10x Genomics platform, the Chromium Next GEM Single Cell 3' Kit v3.1, 16 rxns (PN-1000268), Chromium Next GEM Chip G Single Cell Kit, 48 rxns (PN-1000120), Dual Index Kit TT Set A, 96 rxns (PN-1000215), 3' Feature Barcode Kit (1000262) and Dual Index Kit NT Set A, 96 rxns (PN-1000242) were used. The concentration of each library was accurately determined through qPCR utilizing the KAPA library Quantification Kit according to the manufacturer's protocol (KAPA Biosystems/Roche) to produce cluster counts appropriate for the Illumina NovaSeq6000 instrument. Normalized libraries were sequenced on a NovaSeq6000 S4 Flow Cell using the XP workflow and a 50x10x16x150 sequencing recipe according to manufacturer protocol. A median sequencing depth of 50,000 reads/cell was targeted for each Gene Expression Library and 5000 reads/cell for each Feature Library.