GSM8012315: TOF-012-W16; Homo sapiens; RNA-Seq
Colon biopsies from patients with active UC starting tofacitinib were collected at baseline (n=11) and during follow-up (n=12), placed immediately in cold Hank's Balanced Salt Solution (HBSS) (Gibco, MA, USA) and kept at 4º until processing (<1 h). Freshly collected biopsies were washed with HBSS/DTT (Roche, Spain) 5mM for 15 min and then washed in complete medium (CM) (RPMI 1640 medium (Lonza, MD, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Biosera, France), 100U/ml penicillin, 100 U/ml streptomycin and 250 ng/ml amphotericin B (Lonza), 10µg/ml gentamicin sulfate (Lonza) and 1,5mM Hepes (Lonza) for 10 minutes. Both incubations were performed at room temperature in a platform rocker. Biopsies were chopped with a scalpel and placed into tubes containing 500 μl of Digestion Solution (CM + Liberase TM (0.5 Wünsch units/ml) (Roche, Spain) + DNase I (10 μg/mL) (Roche, Spain) and incubated on a shaking platform for 1h at 250rpm at 37ºC. After incubation biopsies were filtered 2 times through a 50-μm cell strainer and 3 times through a 30-μm cell strainer (CellTrics, Sysmex, USA), washed with Dulbecco's Phosphate Buffered Saline (PBS; Gibco, USA) and resuspended in RPMI (Roswell Park Memorial Institute) medium supplemented with 0.05% of Bovine fetal serum (BSA) at a final concentration of ~500.000-1·106 cells/mL (Veny et al, JCC 2020). Library was performed according to the manufacter's instructions (single cell 3' v3 protocol, 10x Genomics). Briefly, GCs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode.