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| Status |
Public on Oct 04, 2019 |
| Title |
D8 A |
| Sample type |
SRA |
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| Source name |
Boundary organoid
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| Organism |
Homo sapiens |
| Characteristics |
cell type: iPSC derived boundary organoid
region: Anterior part of micro-dissected organoid
culture period: day8 from undifferentiated iPSC
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| Growth protocol |
colonies of hiPSCs were isolated in Accutase and 150,000 cells/mL were plated on Matrigel coated tissue culture plate. Medium was changed to RPMI 1640 medium containing 100 ng/mL Activin A and 50 ng/mL bone morphogenetic protein 4 at Day 1, 100 ng/mL Activin A and 0.2 % fetal calf serum at Day 2 and 100 ng/mL Activin A and 2% FCS at Day 3. For Day 4-7, cells were cultured in gut growth medium (Advanced DMEM/F12 with 15 mM HEPES, 2 mM L-glutamine, penicillin-streptomycin, B27 and N2) supplemented with 200 ng/mL noggin, 500 ng/ml fibroblast growth factor 4 and 2 µM CHIR99021 for anterior gut cell induction and supplemented with 500 ng/ml FGF 4 and 3 µM CHIR99021 for posterior gut cell induction. Cultures for cell differentiation were maintained at 37°C in an atmosphere of 5% CO2 / 95% air and the medium was replaced every day. On Day 7, anterior or posterior gut cells were dissociated to single cells by incubation with TrypLE Express at 37°C. Cells were centrifuged at 1000 rpm for 3 minutes and, after removing supernatant, the pellet was re-suspended in gut growth medium containing 10 uM of Y-27632 dihydrochloride. The anterior or posterior gut cell suspensions were plated on 96 well round bottom ultra-low attachment plate at density of 10,000 cells/well and incubated at 37°C for 24 hours to form spheroid. On Day 8, generated single anterior spheroid and posterior spheroid were mixed on 96 well round bottom ultra-low attachment plate in gut growth medium for 24 hours to form fused boundary spheroids (A-P spheroids). On Day9, A-P spheroids were embedded in Matrigel drop and were cultured in gut growth medium to generate multi-organ organoids.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using the RNeasy mini kit (Qiagen, Hilden, Germany).
Sample preparation for RNA sequencing was performed using SMART-seq v4 Ultra Low Input RNA Kit for Sequencing (Clontech Laboratories) according manufacture’s user manual.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Illumina OLB 1.9.4 software used for basecalling and bcl conversion/demultiplexing/fastq creation.
Transcripts Per Million (TPM) were quantified using kallisto v0.44.0
Supplementary_files_format_and_content: tab-delimited text files include TPM values for each Sample.
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| Submission date |
May 03, 2019 |
| Last update date |
Oct 04, 2019 |
| Contact name |
Norikazu Saiki |
| E-mail(s) |
nrsk.ior@tmd.ac.jp
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| Organization name |
Tokyo Medical and Dental University
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| Department |
Institute of Research
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| Street address |
1-5-45 Yushima, Bunkyo-ku
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| City |
Tokyo |
| ZIP/Postal code |
113-8510 |
| Country |
Japan |
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| Platform ID |
GPL16791 |
| Series (1) |
| GSE121830 |
RNA-sequencing with micro-dissected boundary organoid into anterior, posterior, and boundary regions |
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| Relations |
| BioSample |
SAMN11569573 |
| SRA |
SRX5785474 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3747600_D8A_human37_Gene_TPM.xls.gz |
257.5 Kb |
(ftp)(http) |
XLS |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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