title: Timecourse analysis of human colorectal cancer organoids under FOLFIRI chemotherapy
design: Timecourse analysis of human colorectal cancer organoids under FOLFIRI chemotherapy
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 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 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.  Organoids were seeded as single cells at a density of 20 000 cells per 20 µL dome, counting for a total of 400 000 cells plated per timepoint. After seven days that spheres were generated, FOLFIRI treatment was started at a concentration representative of the IC50 and timepoints were collected at 0, 12, 24, 48, and 72 h treatment. After 72h, FOLFIRI was removed and changed with either fresh medium to let the organoids recover (“Release timeline”), or FOLFIRI was reapplied (“Continuous timeline”). Timepoints in the “Release timeline” were collected after 12, 24, 48 and 72 h, while the “Continuous timeline” included a timepoint collected after 48 h. A maximum of 40 000 cells from each pooled sample was loaded onto a well of a 10X Chromium Next GEM Chip G and scRNA-sequencing libraries were generated following the Chromium Next GEM Single Cell 3' Reagent Kit v3.1 (Dual Index protocol) according to the manufacturer's instructions. Each reaction generated a library for gene expression (GEX) and a library for hashtag oligos (HTO). 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. 