HTO_S01
Homo sapiens
Protocols: 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.
ENA first public
2026-04-01
INSDC center name
DKFZ Heidelberg
INSDC status
public
Submitter Id
E-MTAB-16843:HTO_S01
age
76
broker name
ArrayExpress
cell type
cancer cell
collection date
not collected
common name
human
compound treatment design
multiplexed
developmental stage
adult
disease
colorectal cancer
geographic location (country and/or sea)
not collected
individual
HD42466
isolate
not applicable
organism part
colon
sample name
E-MTAB-16843:HTO_S01
scientific_name
Homo sapiens
sex
male
single cell library information
HTO
time unit
multiplexed