title: Single-cell RNA-sequencing of murine colorectal cancer tumors
design: Single-cell RNA-sequencing of murine colorectal cancer tumors
library_construction_protocol: Organoids were enzymatically dissociated and strained to obtain single cells and frozen until processed for single-cell sequencing. For sample multiplexing, single cells were incubated with hashing antibodies (Total-Seq-B, BioLegend, 1 µg antibody) and additional cell sorting antibodies (EpCAM, Miltenyi) according to the manufacturer's protocol. In short, single cells were thawed and washed in FACS buffer. Cells were resuspended in 50-100 µl with Fc Blocking Reagent Mouse (Miltenyi) in FACS buffer (ratio 1:50) and incubated with the antibody cocktail at 4°C for 30 min in the dark. Then, samples were washed three times in FACS buffer before resuspending in 600 µl FACS buffer with ZombieNIR (BioLegend) for live tumor cell isolation. Hashtag oligo-labelled samples were pooled and processed for single-cell RNA-sequencing using the 10X Chromium Next GEM Single Cell Reagent Kit 3.1 according to the manufacturer's instructions (Dual Index protocol). First, cells are resuspended in a master mix and loaded onto a chip. After encapsulation, cells were lysed inside their GEMs. After cell lysis inside of GEMs, mRNA is reverse transcribed to cDNA with droplet-specific barcodes. After GEM breakdown, the library is being prepared according to the protocol including cDNA amplification by PCR. 