title: Time course analysis of human colorectal cancer organoid treatment with Sacituzumab Govitecan (SG) or untargeted control IgG1-SN-38
design: Time course analysis of human colorectal cancer organoid treatment with Sacituzumab Govitecan (SG) or untargeted control IgG1-SN-38
library_construction_protocol: Organoids were harvested and washed twice with cold PBS (1500 rpm, 4 °C, 5 min). Dissociation of the organoids was achieved resuspending the pellet in TrypLE (Thermo Fisher Scientific, catalog #12605036) and incubated at 37 °C in a water bath until a homogeneous cell suspension was obtained, pipetting the mix occasionally to mechanically accelerate the dissociation process. To stop the reaction, PBS + 2% FCS was added to the solution and spin down (1 500 rpm, 4 °C, 5 min). Eventually, cells were cryopreserved until all timepoints were collected for further processing.  For sample multiplexing, frozen samples were rapidly thawed in a water bath 37 °C and transferred to a conical tube with added fresh organoid medium + 2% FBS and spin down. Cell pellet was then resuspended in 100 µL of PBS + 2% FCS with 1:50 FcR Blocking Reagent (Miltenyi Biotec, catalog #130-059-901) and stained with 1µL of hashing antibodies (Total-Seq-B, BioLegend, 0.5 µg antibody) for 30 minutes at 4 °C. Afterwards, cells were washed twice with PBS + 2% FCS. Cell pellet was then resuspended in PBS + 2% FCS added with a live/dead marker (DAPI, Invitrogen catalog # 62248 or Zombie NIR, BioLegend, catalog # 423106), filtered, and multiplexed samples were sorted for live cells in a single tube.  Hashtag oligo-labelled samples were pooled (up to 12,000 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. 