GSM7073191: Paclitaxel-treated, scRNAseq; Homo sapiens; RNA-Seq
Tumors were resected and stored in cold DMEM media on ice for transport. After necrotic tissue was removed using a scalpel, the samples were minced and dissociated in a gengleMACS Octo (Miltenyi, 130-096-427) to single-cell suspension using a human tumor dissociation kit (Miltenyi, 130-095-929) at 37C per the manufacturer's instructions with the following change: samples were checked after 30 min and the protocol was terminated if chunks of undissociated tissue were not visible. If a significant amount of undissociated sample remained, the program was run for 15min additional time. After dissociation, the samples were filtered through a 100uM strainer, the cells pelleted, the supernatant removed, and red blood cells were lysed using RBC lysis buffer (Invitrogen, 50-112-9751). RBC lysis buffer was diluted in DMEM, and the cells were washed prior to mouse cell depletion using magnetic beads (Miltenyi, 130-104-694). The resulting suspension of human tumor cells was adjusted to a concentration appropriate for loading into a 10X genomics Chromium controller (3' gene expression kit, #120267) with an expected output of 7,000 cells/sample. Samples were run in separate wells and libraries constructed according to the manufacturer's instructions. Libraries were sequenced on an Illumina Novaseq 6000, aiming for approximately 100,000 reads/cell.