title: GSM8702778: Tumoroids, HuCo021320-P10; Homo sapiens; RNA-Seq
library_construction_protocol: Dissociated tumor cells were purchased from Discovery Life Sciences. Vials of cryopreserved HuCo021320 and HuCo3209 were used for the experiment. Tumoroid lines were derived from one of the original DTC vials and were expanded in OncoPro media. Cell banks were generated throughout the culture by resuspending dissociated tumoroids at 2×10^6 cells/ml in Gibco™ Recovery™ Cell Culture Freezing Medium (Thermo Fisher Scientific, cat# 12648-010), freezing overnight at -80°C in a Mr. Frosty™ Freezing Container (Thermo Scientific™, 5100-001), and storing in a liquid nitrogen dewar. Cryopreserved HuCo021320 at passages 10 and 27, and cryopreserved HuCo3209 at passage 10, were used for single-cell RNA sequencing experiments. Cryopreserved single cells from dissociated colorectal tumors and tumoroids were submitted to Azenta Life Sciences (Burlington, MA, USA) for scRNA-seq. The sample libraries were prepared in a single batch using the Chromium Single-Cell 3′ v3 Library and Gel Bead Kit (10x Genomics). Briefly, gel bead-based emulsions (GEM) were generated by combining barcoded single-cell 3′ Gel Beads, cells, and partitioning oil. Ten times barcoded, full-length cDNAs generated from GEMs were amplified by polymerase chain reaction (PCR). Enriched libraries were enzymatically digested, size selected, and adaptor ligated for sequencing. Sequencing libraries were generated with unique sample indices for each sample and quantified using the Kapa library kit. Quantified libraries were sequenced on an Illumina NovaSeq™ 6000.