title: GSM8545007: 931-TKA-TGFb__0h; Mus musculus; RNA-Seq
library_construction_protocol: After mechanical disruption of 931-TKA organoids, the resulting organoid fragments were seeded in 3 mg/ml Matrigel and cultivated until they had re-formed cystic structures. Following this, organoids were treated with solvent or TGF-β1 for up to 72 h. Post-treatment, organoids were dissociated into single cells using TrypLETM Express. The resultant single organoid cells were washed once with PBS and resuspended in PBS/0.04% bovine serum albumin to enhance cell viability. Larger organoid fragments and cell debris were removed with a 30 µ MACS SmartStrainer (Miltenyi Biotec B.V. & Co. KG, Bergisch Gladbach, Germany). A BD FACSAria™ Fusion flow cytometer was then used to enrich for viable single cells. Organoid single cells were mixed with reverse transcriptase (RT) , RT reagent, and template switch oligos, following the Chromium Next GEM Training Kit User Guide (v3.1, 10x Genomics). In brief, the cell-emzyme mixtures were combined with master mix, partitioning oil, and gel beads, then loaded into a Chromium chip and processed in the 10x Chromium controller to form gel beads in emulsion (GEMs). In GEMs, reverse transcription was performed, followed by cDNA amplification and clean-up steps. Adhering strictly to the 10x Genomics user guide, sequencing libraries were prepared, followed by SPRIselect bead cleanups. Finally, libraries from solvent and TGF-β1-treated organoid cells were pooled at a final concentration of 10 nM for sequencing.