GSM8323261: Samples 4-5, PBMC, post treatment, Adalimumab non-responder (CMO); Homo sapiens; RNA-Seq
Peripheral venous blood was collected into K2EDTA tubes (Vacutainer BD, Franklin Lakes, NJ, USA). The PBMCs were isolated using density gradient centrifugation with Lympholyte H Cell Separation Medium (Cedarlane, Burlington, ON, Canada) and SepMate 50 centrifuge tubes (StemCells Technologies, Vancouver, BC, Canada). The remaining platelets and erythrocytes were removed using the low-speed centrifugation and red blood cell lysis, respectively. The isolated PBMCs were stored in liquid nitrogen at a density of 1-10E6 cells/mL. A combination of 10% dimethyl sulfoxide (Sigma-Aldrich, Bur-lington, MA, USA) and 40% heat-inactivated fetal bovine serum (Gibco, ThermoFisher Scientific, Waltham, MA, USA) was used as the cryopreservation medium. The cell concentration was determined using the CytoSmart automated cell counter (Corning Inc., Corning, NY, USA). To perform scRNA-seq the 10X Chromium System (10X Genomics, Pleasanton, CA, USA) was used. First, the cryopreserved PBMCs were thawed and washed in Dulbecco's Phosphate-Buffered Saline supplemented with 5% FBS to remove the cryopreservation medium. The quality of the cell samples was confirmed using the trypan blue exclusion and viability of all samples was >90%. 2E6 cells per sample were used for the library preparation. Samples 001 and 002 were processed as single reactions using Chromium Next Gem Single Cell 3' Kit v3.1 (PN-1000269), Chromium Next GEM Chip G Single Cell Kit (PN-1000127), and Dual Index Kit TT Set A (PN-1000215; all 10X Genomics) according to the manufacturer's instructions (CG000315 Rev E). Other samples were processed as multiplexed reactions, with three samples per reaction, using Chromium Next Gem Single Cell 3' Kit v3.1 (PN-1000269), 3' Feature Barcode Kit (PN-1000262), Dual Index Kit TT Set A (PN-1000215) and Dual Index Kit NN Set A (PN-1000243; all 10X Genomics). For the multiplexed samples, the cells were first CMO labelled using 3'CellPlex Kit Set A (PN-1000261, 10X Genomics) according to the manufacturer's instructions (CG000391 Rev B). All samples were loaded onto the Chromium Controller Instrument (10X Ge-nomics) to generate gel bead-in-emulsions (GEM). Next, complementary DNA (cDNA) libraries were constructed. GEM–reverse transcription (RT) was performed in a C1000 deepwell block thermocycler (BioRad, Hercules, CA, USA). The cDNA was purified using Dynabeads MyOne Silane Beads (Thermo Fisher Scientific) and amplified following the recommendations in the 10X Genomics protocol CG000315 Rev E, for single reaction samples, and protocol CG000388 Rev C, for multiplex reaction samples. The product was subsequently purified using the SPRIselect Reagent Kit (B23318, Beckman Coulter, Brea, CA, USA,). Next, 3' Gene Expression Libraries were constructed using the Chromium Single-Cell 3′ Library Construction Kit (PN-1000196). For the multiplexed samples Cell Multiplexing Libraries were additionally generated using Dual Index Kit NN Set A (PN-1000243) and 3'Feature Barcode Kit (PN-1000262). Library quality control was performed using the Agilent Bioanalyzer 2100 and using High Sensitivity DNA Kit (Agilent, Santa Clara, CA, USA). The prepared libraries were sequenced with Illumina NovaSeq pair-end 150bp at GENEWIZ (GENEWIZ Germany GmbH, Leipzig, Germany) with an average sequencing depth of >100 million reads per library yielding approximate average of 28,600 reads per cell.