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| Status |
Public on Feb 23, 2021 |
| Title |
Microwell Round_Day40_Rep2 |
| Sample type |
SRA |
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| Source name |
Human cerebral organoids
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| Organism |
Homo sapiens |
| Characteristics |
cell line: H9 human embryonic stem cells (WA09, WiCell)
time point: Day 40
embryoid body formation method: Agarose microwell shape round
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| Growth protocol |
Feeder-independent H9 human embryonic stem cells (hESCs) (WA09) were obtained from WiCell. Cells were maintained in tissue culture dishes (Fisher Scientific Corning Costar) coated with 0.5 mg/cm2 Vitronectin (VTN-N) (Thermo Fisher Scientific) in E8 medium (Thermo Fisher Scientific) and passaged using standard protocols. 96-well and microwell human cerebral organoids (hCOs) were generated using the same pool of hESCs and differentiated using the same protocol as previously described (Lancaster et al. 2013, Nature). Number of cells seeded per well was experimentally optimized for successful generation of embryos bodies (EB) in microwells. For each microwell device, 60 µL of hESC suspension at 32,000 cells/well was pipetted into the microwell enclosure using a cut p200 tip. The device was gently placed into the incubator for 5 minutes to allow cells to settle into the microwells. After 5 minutes, 2.5 mL media was slowly added to the wells avoiding the disruption of the seeded cells in the microwells. Media was changed every 12 hours for the first 3 days with fresh hESC medium with basic fibroblast growth factor (bFGF) (Invitrogen) and ROCK inhibitor (Y-27632, LC laboratories) for all hCOs (microwell and 96-well). All cells and hCOs were maintained in a humid incubator at 37 °C with 5 % CO2. After 5 days all hCOs were transferred into low-attachment 24-wells plates (1 hCO/well) and the remainder of the previously described whole brain organoid protocol was followed (Lancaster et al. 2013, Nature).
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| Extracted molecule |
total RNA |
| Extraction protocol |
For total RNA extraction the following samples were collected from three independent culture plates: 10 pooled human cerebral organids (hCO) for day 10 samples, 6 pooled hCOs for day 20 samples, and 4 pooled hCOs for day 40 samples. hCOs were washed 3 times in cold PBS. Matrigel was dissolved by incubating the hCOs in chilled Cell Recovery Solution (Corning, cat. no. 354253) for 1h at 4 °C. The dissolved Matrigel was removed by rinsing 3 times in cold PBS. Total RNA was isolated using Direct-zol RNA MicroPrep Kit (Zymo Research) according to the manufacturer’s protocol. RNA samples were collected in 2mL RNAse-free tubes and chilled on ice throughout the procedure.
RNA libraries were prepared and sequenced by BGI using DNB-seq PE100 platform.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
DNBSEQ-G400 |
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| Data processing |
Raw FASTQ formatted sequence reads were imported into CLC Genomics Workbench (version 20.0.2, QIAGEN Digital Insights)
Adaptor sequences and bases with low quality were trimmed and reads were mapped to the reference genome (GRCh38.102) using the RNAseq analysis tool with the default parameters recommended for RNAseq analysis in CLC Genomics Workbench (version 20.0.2, QIAGEN Digital Insights).
Gene expression is estimated with the EM estimation algorithm and reported as TPM (Transcripts Per Million) and RPKM (Reads per kilobase of transcript per million mapped reads) using CLC Genomics Workbench (version 20.0.2, QIAGEN Digital Insights).
Genome_build: GRCh38.102
Supplementary_files_format_and_content: All process data files are .xlsx files including name, chromosome, region, expression value, TPM, RPKM, exons, gene ID, gene length, ENSEMBL ID, biotype, unique reads, total reads, annotated transcripts, uniquely identified transcripts, exon length, unique exon reads, total exon reads, ratio of unique to total exon reads, unique exon-exon reads, total exon-exon reads, unique intron reads, total intron reads and ratio of intron to total gene reads for each gene.
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| Submission date |
Feb 22, 2021 |
| Last update date |
Feb 23, 2021 |
| Contact name |
Albert J Keung |
| E-mail(s) |
ajkeung@ncsu.edu
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| Organization name |
North Carolina State University
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| Department |
Chemical and Biomolecular Engineering
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| Street address |
911 Partners Way
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| City |
Raleigh |
| State/province |
NC |
| ZIP/Postal code |
27606 |
| Country |
USA |
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| Platform ID |
GPL28038 |
| Series (1) |
| GSE167208 |
Effects of early geometric confinement on the transcriptomic profile of human cerebral organoids |
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| Relations |
| BioSample |
SAMN18025367 |
| SRA |
SRX10149165 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5099584_59_1_GE_.xlsx |
3.3 Mb |
(ftp)(http) |
XLSX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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