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| Status |
Public on Jun 01, 2020 |
| Title |
P1_06222016 |
| Sample type |
SRA |
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| Source name |
PDAC derived organoid
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| Organism |
Homo sapiens |
| Characteristics |
patient id: Patient 1
tissue: epithelial
Stage: mature
cell type: PDAC derived organoid
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| Growth protocol |
To establish organoid cultures, a piece of subcutaneous PDX tumors was extracted, minced and digested with collagenase type XI and dispase in DMEM and incubated from 0.5-1 h at 37 ºC with gentle shaking. Cells were spin and dissociated with TrypLE Express and DNase I for 10 min at 37 ºC, and washed with DMEM. For culturing organoids, dissociated cells were washed and embedded in growth-factor-reduced (GFR) Matrigel and cultured in complete media (Intesticult, A83-01 0.5 µM, fibroblast growth factor 10 100 ng/ml, Gastrin I 10 nM, N-acetyl-L-cysteine 10 mM. Nicotinamide 10 mM, B27 supplement 1x, Primocin 1 mg/ml and Y-27632 10.5 µM).
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| Extracted molecule |
total RNA |
| Extraction protocol |
The single cell RNA-seq dataset is comprised of 15 separate drop-seq runs. For each run, a total of 2000 fully growth organoids were pooled, typsinized with TrypLE in presence of 10 µM Y-27632, strained into a single cell suspension and counted. The final concentration of the cell suspension varied between 100-120 cells/uL depending on the flow rate at which monodisperse droplets were formed. Organoids were always harvested for drop-seq analysis at same growth size to ensure uniformity from run to run.
The droplets were broken and the beads were collected and reverse-transcribed. The cDNA obtained was then PCR amplified and quantified. Finally, the cDNA was fragmented
and amplified using primers that
allow amplification of only the 3’
ends, processed into RNA-seq
libraries using Illumina Nextera XT library prep kit.
As described in Macosko et al Cell (2015)
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Fastq files are trimmed if needed, such that read1=20bp, read2=50bp;Unaligned BAM file created using picard-tools FastqToSam
Drop-seq_alignment.sh (provided by Steve McCarroll lab) is started, which tags cell and molecular barcodes, trims adaptor and polyA sequences, performs alignment, adds gene annotations
DetectBeadSynthesisErrors.sh (provided by Steve McCarroll lab) is run with NUM_BARCODES=2x expected no of cells, then DGE matrix generated with MIN_NUM_GENES_PER_CELL=250
DGEs for all runs are merged, then normalized, scaled and log transformed. Cell cycle and batch effects are regressed out.Training set created, highly variable gene identified.
A topic model is generated from the training set using the highly variable genes as the vocabulary
a topic distribution is calculated for the cells not included in the training set and cells are assigned to the topic with the highest probability, and clusters with fewer than 50 cells are removed
Pairwise marker genes for each cluster are identified using genes that are expressed in least 10% of the cells in both clusters and have average log fold change >1 between clusters. Clusters that do not have at least 10 unique marker genes are merged
Genome_build: hg19
Supplementary_files_format_and_content: text files
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| Submission date |
May 15, 2018 |
| Last update date |
Jun 01, 2020 |
| Contact name |
Miguel Brown |
| E-mail(s) |
miguel.a.brown@gmail.com
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| Organization name |
The Rockefeller University
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| Street address |
1230 York Ave Box 186
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| City |
New York |
| State/province |
New York |
| ZIP/Postal code |
10065 |
| Country |
USA |
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| Platform ID |
GPL18573 |
| Series (2) |
| GSE114454 |
Organoids derived from pancreatic adenocarcinoma patients share morphological and genetic features with the primary tumor [Drop-seq] |
| GSE114455 |
Organoids derived from pancreatic adenocarcinoma patients share morphological and genetic features with the primary tumor |
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| Relations |
| BioSample |
SAMN09209632 |
| SRA |
SRX4083307 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3142043_P1_06222016_total-min250genes.dge.txt.gz |
532.4 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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