GSM8898806: ccRCC tumor, untreated, sc03, far; Homo sapiens; RNA-Seq
Tumors were coarsely minced using a scalpel and enzymatically dissociated using the human tumor dissociation kit (Miltenyi) for 25mins at 37c using the Octo-dissociator (Miltenyi). Digested cells were neutralized in DMEM + 10% FBS and successively washed in PBS and strained through 100um, 70μm and 45μm cell strainers with an RBC lysis step in between (Ack lysis buffer; Gibco). Single cell suspensions were stained with DAPI (BD Pharmingen) and calcein AM (Invitrogen) and FACS sorted for live single cells. Cells were counted using a hemocytometer and up to 10,000 cells were loaded into the Chromium Controller (10x genomics). Cells were processed using the Next GEM 5' mRNA V2 kit (10x genomics) with strict adherence to manufacturer's protocols. Gene expression libraries were sequenced at a target depth of 200 million reads per sample.