|
| Status |
Public on Apr 01, 2024 |
| Title |
ONS76, tumour-spheroid, replicate 1 |
| Sample type |
SRA |
| |
|
| Source name |
ONS-76 tumour-spheroid
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: ONS-76 tumour-spheroid
cell line: ONS-76
cell type: cell line
genotype: malignant
treatment: in vitro culture
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Day 60 organoids and organoid-tumour co-culture samples were collected using a wide bore pipette tip and placed in a 6 well plate. Using micro-scissors, organoids were cut in four pieces before placing a total of five organoids in an Eppendorf tube. After removal of excess medium, 200 uL of pre-warmed (to 37°C) Neuron isolation enzyme was added and the tube was placed in a 37°C water bath. Organoids were incubated with the enzyme for 30 minutes with intermittent agitation. The enzyme was removed and organoids were washed once with HHGN medium (HBSS + 2.5mM HEPES + 35mM Glucose + 4mM NaHCO3). Organoids were then dissociated in differentiation medium by gentle trituration. Afterwards, the cell suspension was enriched for viable cells using the dead cell removal kit (Miltenyi Biotec) following the manufacturers protocol. Cell viability was determined using Trypan Blue staining and counted using an automated cell counter (Applied Biosystems Countess 3). Cells were centrifuged at 300 x g for 5 minutes and resuspended in the appropriate volume of resuspension buffer (HBSS + 0.04% BSA) to a final concentration of 2 x 10^3 cells / uL before proceeding with single cell RNA sequencing. Thirty tumour spheroids were collected using a wide bore pipette tip and placed in an Eppendorf tube. Excess medium was removed and 200 uL of pre-warmed Neuron isolation enzyme (Gibco) was added. The tube was placed in a 37°C water bath for 20 minutes and agitated every 5 minutes. Subsequent processing was performed following the same steps as described above. DAOY and ONS-76 cell monolayer cultures were dissociated by incubation with pre-warmed TrypLE for 5 minutes. TrypLE was then diluted in PBS before centrifugation at 400 x g for 5 minutes to collect the cells. Cells were washed once in differentiation medium before processing as described above.
Single cell barcoding and reverse transcription of poly-A mRNA was performed using the 10X Chromium controller (3’ version 3.1 chemistry) and sequencing of single cell libraries was performed on an Illumina NovaSeq6000 sequencing system according to the manufacturer’s instructions
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
10X Genomics 3pr v3.1 Chemistry
03_ONS76_SPHER_GEX-v3.1
|
| Data processing |
Analysis pipeline was applied to process Chromium single-cell data to align reads, and generate feature-barcode matrices. Illumina’s bcl2fastq and cellranger mkfastq demultiplexes were used to convert the raw base call (BCL) files generated by Illumina sequencers into FASTQ files.
Cellranger count (v7.0.0) took FASTQ files from cellranger mkfastq and performed alignment, filtering, barcode counting, and UMI counting. It used the Chromium cellular barcodes to generate feature-barcode matrices. In the count pipeline, one sample was processed through one GEM well and sequenced on one flowcell.
Assembly: GRCh38
Supplementary files format and content: Seurat object (counts of all samples)
|
| |
|
| Submission date |
Feb 02, 2024 |
| Last update date |
Apr 01, 2024 |
| Contact name |
John Jacob |
| Organization name |
University of Oxford
|
| Department |
NDCN
|
| Street address |
Headley Way
|
| City |
Oxford |
| ZIP/Postal code |
OX3 9DU |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL24676 |
| Series (1) |
| GSE254917 |
Sonic hedgehog medulloblastoma cells in co-culture with cerebellar organoids converge towards in vivo malignant cell states |
|
| Relations |
| BioSample |
SAMN39748475 |
| SRA |
SRX23508714 |
