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| Status |
Public on Apr 06, 2023 |
| Title |
AG c15 Attach 5 days |
| Sample type |
SRA |
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| Source name |
AG05836B C15
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| Organism |
Homo sapiens |
| Characteristics |
cell line: AG05836B C15
cell type: Neuron
genotype: Ctrl
treatment: Neural differentiation
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| Treatment protocol |
Differentiation of hiPSC towards neural lineage was performed.Briefly, iPSCs were pretreated with 50 μM Y-27632 (Stemcell Technologies) for 1 h. Then the cells were dissociated with Accutase (Stemcell Technologies) and plated onto low attachment V-type 96-well plate at 20 000 cells per well or AggreWell800 plate (Stemcell Technologies) to form aggregates. The culture was maintained in neural induction medium consisting of DMEM/F12 supplemented with 2% B27 supplement without vitamin A, 1% N2 supplement, 200 nM Noggin (R&D Systems) and 10 μM Y-27632 for 48 h. Formed aggregates were then transferred onto low attachment 6-well plates and continued culture for another 4 days. To induce neural rosette formation, the aggregates were transferred onto Geltrex coated 6-well plate and cultured for 4 days in neural induction medium supplemented with 200 ng/mL Noggin, 200 ng/mL DKK1 (R&D Systems), and 20 ng/mL bFGF (Life Technologies). Neural rosettes were manually picked and transferred onto low attachment 6-well plate in neural induction medium supplemented with 10 ng/mL bFGF and 10 ng/mL EGF to allow neuroepithelial sphere formation. Five days later, two procedures were performed to directly neural differentiation. First, the neural aggregates were placed onto Geltrex coated plates and treated with neural differentiation medium consisting of Neurobasal A medium supplemented with 10 ng/mL BDNF, 10 ng/mL GDNF, 200 μM ascorbic acid, 500 μM cyclic AMP. Second, the neural aggregates were dissociated into single cells with Accutase and replated onto poly-D-lysine/laminin coated 24 or 48-well plates for further differentiation with neural differentiation medium supplemented with 10 ng/mL BDNF, 10 ng/mL GDNF, 200 μM ascorbic acid, 500 μM cyclic AMP.
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| Growth protocol |
The iPSCs were continuously cultured and expanded on Geltrex coated 6-well plates with Essential 8 medium (ThermoFisher Scientific, Cat#A1517001).
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from patient and control samples using the RNeasy Kit (Qiagen) following the manufacture’s protocol. The quality of purified RNA was estimated on an Epoch Microplate Spectrophotometer (Bio-Tek).
Total RNA was isolated from patient and control samples using the RNeasy Kit (Qiagen) following the manufacture’s protocol. The quality of purified RNA was estimated on an Epoch Microplate Spectrophotometer (Bio-Tek).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
neu3.wt
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| Data processing |
Sequence reads were checked for adaptor sequence/low-quality sequence
Sequence reads were mapped to GRCh38/hg38 using hisat2
Read count extraction was performed using featureCounts
Downstream DEGs analysis was performed with DESeq2 in R
Pathway and GO analysis was performed with GSEA
Assembly: GRCh38/hg38
Supplementary files format and content: tab-delimited text file including raw counts of sequencing reads for the features of interest and for all samples in the same matrix
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| Submission date |
Jun 21, 2022 |
| Last update date |
Apr 06, 2023 |
| Contact name |
Magnar Bjørås |
| E-mail(s) |
magnar.bjoras@ntnu.no
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| Organization name |
Faculty of Medicine and Health Sciences,NTNU
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| Department |
Department of Clinical and Molecular Medicine (IKOM)
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| Lab |
M.Bjørås´s Lab
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| Street address |
Erling Skjalgssonsgate 1
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| City |
Trondheim |
| ZIP/Postal code |
7030 |
| Country |
Norway |
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| Platform ID |
GPL16791 |
| Series (1) |
| GSE206614 |
A loss-of-function mutation in human Oxidation Resistance 1 disrupts the spatial-temporal regulation of histone arginine methylation in neurodevelopment |
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| Relations |
| BioSample |
SAMN29229944 |
| SRA |
SRX15819310 |
| 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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