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| Status |
Public on May 05, 2022 |
| Title |
S1_T_O_rep1 |
| Sample type |
SRA |
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| Source name |
Patient 1 normal derived organoid
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| Organism |
Homo sapiens |
| Characteristics |
patient id: Patient 1
patient diagnosis: colorectal cancer
tissue: normal derived organoid
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| Treatment protocol |
The tumor and adjacent normal colon tissue that derived from biopsies of CRC patients were isolated and dissected into small pieces using surgical scissors separately. After washed by cold PBS at least three times, the tissue was further digested at 37℃ for about 40 to 60 min with 2mg/ml Collagenase/Dispase(Roche). The digested cell suspension was then collected by centrifuging at 800g for 5min and re-suspended. As for the organoid cultures, each cell cluster was picked out and digested in a about 200μl drop of TrypLE Express (Invitrogen) on a 3.5mm dish at 37℃ for 20-30 min, which were prepared for further cell-picking.
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| Growth protocol |
Organoid culture was performed as previously described (Chua et al., 2014), with minor modifications to the tissue dissociation protocol to improve cell viability. In brief, crypts from normal tissues were isolated through gently scraping from washed tissues. Then crypts were mixed with Matrigel (Biosciences) and cultured in 24-well plates (50μL Matrigel/well). As for tumor tissues, the supernatant contained cells were first centrifuged at 400g for 5 minutes at 4°C. Then the cell pellet was resuspend with Matrigel and cultured in 24-well plates (50μL Matrigel/well).The composition of normal colonic organoid culture medium:Advanced DMEM/F12 , 100 U/ml penicillin/streptomycin, 2 mM GlutaMAX, 10 mM HEPES, 0.5 μM A83-01 (Tocris), 1x B27 (Invitrogen), 3 μM SB202190 (SIGMA), 10 nM prostaglandin E2(SIGMA), 0.5μg/ml R-spondin(Nuvelo) , 10mM nicotinamide (SIGMA), 1 mM N-acetylcysteine (Sigma) , 10 nM gastrin I (Sigma),50 ng/ml EGF (Invitrogen), 100 ng/ml Noggin (Peprotech), 100 ng/mlWnt-3A(Millipore) .10μM Y-27632(Tocris BioScience) was supplemented to the medium in the first week. Tumor organoid medium contain all of the above without Wnt-3A. Medium were changed every 2 days.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Single cells were manually-picked into lysis buffer of single cell RNA-seq carefully. Single cell RNA was released by vortexed for about 45 secs and further incubated at 72℃ for 3 min, which were further proceed to transcription process.
Single cell RNA sequencing protocol: Single cell RNA-seq libraries were prepared using a method developed based on the STRT-seq. The primer designed for transcription was modified: 8bp barcodes was added to label different cells, 8bp UMIs were used to eliminate duplication. After total 19 cycles of PCR amplification, about 300ng of cDNA was acoustic sheared into 300bp. Dynabeads MyOne Streptavidin C1 beads were further used to capture the biotin modified cDNA. Kapa Hyper Prep Kit (Kapa Biosystems) were applied for libraries construction. The libraries were sequenced on Illumina HiSeq 4000.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
Colon_Cancer_Organoid_TPM.txt
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| Data processing |
For data from Illumina, Illumina CASAVA version 1.8 were used to the basecalling
scRNA-Seq:Read 2 was used to abtain the cell barcodes to further split the reads according to the cell IDs(barcode) and the same time recorded the UMI sequences;Read 1 was picked in each cell according to the cell ID in read2 and the UMI information was aligned to it,and then these raw reads were trimmed to remove TSO or polyA sequence;Adaptor contamination and low-quality reads were discarded from the trimmed Read 1 raw data;TopHat(version 2.0.14) with default settings were used for sequence alignment and uniquely mapped reads were kept.
Genome_build: Reference sequence and transcript annotation files were from UCSC genome assembly hg19
Supplementary_files_format_and_content: tab-delimited text files include TPM values for each single cell RNA samples
Supplementary_files_format_and_content: *Info.txt files include the barcode sequence for each cell ID.
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| Submission date |
Sep 17, 2018 |
| Last update date |
May 05, 2022 |
| Contact name |
Rui WANG |
| E-mail(s) |
fish_cat_wr@sina.cn
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| Phone |
15801166445
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| Organization name |
Peking University
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| Department |
Biodynamics Optical Imaging Center (BIOPIC)
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| Lab |
Fuchou Tang
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| Street address |
No.5 Yiheyuan Road, Haidian District
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| City |
Beijing |
| ZIP/Postal code |
100871 |
| Country |
China |
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| Platform ID |
GPL20301 |
| Series (2) |
| GSE120065 |
A comprehensive transcriptome portrait of colorectal cancer derived organoid at single cell levels (Single cell RNA-seq) |
| GSE121455 |
A comprehensive transcriptome portrait of colorectal cancer derived organoid at single cell levels |
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| Relations |
| BioSample |
SAMN10079330 |
| SRA |
SRX4704800 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3392818_S1_T_O_rep1.Info.txt.gz |
602 b |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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