title: Single-cell RNA-sequencing of PDOX colorectal tumors
design: Single-cell RNA-sequencing of PDOX colorectal tumors
library_construction_protocol: Patient-derived tumor organoids were injected into the left flank of mice. Once grown to 1.5 cm, tumors were taken out for analysis. Tumors were enzymatically dissociated with a tumor dissociation kit (Miltenyi) following the manufacturer's instructions. In short, tumor fragments were incubated together with an enzyme cocktail in a gentleMACS Octo Dissociator with heaters (Miltenyi) using the default programs 37C_h_TDK_1 (for human tissue). Afterwards, the sample was 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 (anti-human EpCAM, Miltenyi; anti-mouse CD45, BioLegend) 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 (EpCAM-positive, CD45-negative).  Hashtag oligo-labelled samples were pooled (up to 10,000 cells each) 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 in 11 cycles. 