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| Status |
Public on May 05, 2016 |
| Title |
1 month organoid 1 |
| Sample type |
SRA |
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| Source name |
1 month organoid
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| Organism |
Homo sapiens |
| Characteristics |
cell type: hESC-derived cerebral organoids
age: 1 month old
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| Growth protocol |
H9 human embryonic stem cell(hESCs) (WA09) from WiCell was cultured on a feeder layer of irradiated mouse embryonic fibroblasts following previously established protocols. H9 hESCs were detached from their feeder layer using 1mg/ml collagenase for 15-20 minutes and 0.5mg/ml dispase for an additional 15 minutes. Wells were washed with media to collect floating undifferentiated hESCs and colonies were dissociated using Accumax at 37°C for 10 minutes to generate a single cell suspension. At day 0, embryoid bodies were formed using the hanging drop method with 4500 cells/drop in DMEM/F12 media supplemented with 20% knockout serum replacement, 4ng/ml bFGF, NEAA and glutamine. After 2 days of hanging drop culture, embryoid bodies were transferred to sterile petri dishes with refreshed media. After 6 days in culture, embryoid bodies were transferred to new petri dishes containing neural induction media consisting of DMEM/F12, 1:100 N2 supplement, NEAA, glutamine and 1ug/ml heparin until day 11. At day 11, organoids were transferred to Matrigel droplets and cultured in 1:1 mixture of DMEM/F12 and Neurobasal medium supplemented with 1:100 B27 without vitamin A, 1:200 N2, NEAA, insulin, beta-mercaptoethanol and glutamine. Organoids were then transferred to stir flask bioreactors for long term growth on day 15 in the same differentiation media except with the addition of retinoic acid and vitamin A. Media was changed every 3 days.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNAs were collected using Qiagen RNeasy MiniKit following manufacturer's instructions
RNA libraries were prepared for sequencing using standard Illumina protocols
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Group 2
4_S4_R1_001.sorted
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| Data processing |
BWA alignment to hg19
mRNA quantification and annotatino Ensembl in Partek Genomic Suite 6.6
Filtering RPKM for all samples <=0.5, data transformation (log2+1), normalization (quantile normalization)
Differential expression analysis (ANOVA) abs>=2, FDR adjusted P-value<=0.05
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text files include RPKM values for each Sample, fold change, p-value, etc
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| Submission date |
Apr 14, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Tariq Rana |
| E-mail(s) |
trana@ucsd.edu
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| Organization name |
University of California, San Diego
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| Department |
Pediatrics
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| Street address |
9500 Gilman Dr.
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| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92093 |
| Country |
USA |
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| Platform ID |
GPL18573 |
| Series (1) |
| GSE80264 |
Transcriptome analysis of H9 hESC derived cerebral organoids |
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| Relations |
| BioSample |
SAMN04851428 |
| SRA |
SRX1704502 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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