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| Status |
Public on Mar 15, 2023 |
| Title |
RNA_Day39,rep2 |
| Sample type |
SRA |
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| Source name |
Mature interneuron-like cells
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| Organism |
Homo sapiens |
| Characteristics |
cell type: Mature interneuron-like cells
time: Day 39
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| Growth protocol |
We used H9.1 human pluripotent stem cells (hPSCs) that are grown on inactivated mouse embryonic fibroblasts (MEFs) in 6 well plate supplemented with human PSC medium (hPSCM) composed of DMEM/F12 (Gibco cat. 11330, USA), 1% non-essential amino acids (Gibco cat. 11140, USA), 1% GlutaMAX (Gibco cat. 35050, USA), 2-mercaptoethanol (Sigma-Aldrich cat.M3148, USA), knockout serum replacement (Gibco cat. 10828, USA) with 4ng/ml Fibroblast growth factor 2 (FGF2) (Invitrogen cat.13256-029, USA). The hPSCs first are induced into neural stem cells in the first 10 days then the cells are patterned into ventral forebrain progenitors with MGE characteristic. The hPSC are dissociated using TrypLE (Gibco cat. 12604021, USA.) and transfer to 10mm plate in suspension and grown hPSCM without Fibroblast growth factor 2 (FGF2) at the first 24h 10uM rock inhibitor (y27632, STEMCELL cat. 129830-38-2, canada) was used, the two third of medium was removed and replaced by new medium every day. The hPSC are becoming embryonic bodies (EBs) at the first day which are spheres that are in suspension. At day 4, the medium was aspirated and replaced with neural induction medium (NIM) that contain DMEM/F12, 1% non-essential amino acids, 1% N-2 supplement (Gibco cat. 17502-048, USA) and heparin (Sigma-Aldrich cat.H3149, USA) and was incubated additional three days while replacing the medium every other day. At day 7, the EBs are plated in 6 well plate that is coated with laminin (Invitrogen cat.23017-015, USA) in order to facilitate the attachment of the EBs, 35–50 EBs per well were plated, NIM was replaced every other day. Neural rosettes appear at day 10, which is the end of the first stage. Neural rosettes were cultured with NIM containing 1.5 μM Pur (SHH agonists) (StemGent cat.04-0009, USA) and was replaced every other. At day 16, the colonies of the neural rosettes were gently detached and moved to 10 mm plate, the lifted neural rosettes were cultured with NIM containing Pur and B27(Gibco cat. 12587-010, USA) the medium was changed every other day till day 25 which is the end of the second stage. On day 26, MGE progenitor spheres were collected and gently pipetted for 3-4 times to disassociate the cells. Then, the cells were plated on poly-l-ornithine (PLO) (Sigma-Aldrich cat.P3655,USA)and laminin-coated coverslips. MGE cells usually attach to the coverslip after 3–4 h. After 4 h neural differentiation medium (NDM) comprised of neuralbasal medium (Gibco cat. 21103, USA) 1% non-essential amino acids, 1% N-2 supplement containing 1 μM cAMP(Sigma-Aldrich cat.D-0260,USA, 10 ng ml−1 BDNF (Gibco cat. PHC7074, USA), 10 ng ml−1 GDNF (Gibco cat. PHC7044, USA), 10 ng/ml IGF1 (PeproTech cat.100-11, USA) and compound E (EMD Biosciences cat. 565790-500UG) was added to each well. The medium was replaced every 3 days, but without adding compound E which is only added once in order to synchronize cell differentiation. Then, the cells are differentiated and cultured till day 55. In the third stage the progenitors are differentiated to GABAergic-like interneurons by day 39. In long-term culture at day 55 we can get population of neurons that expressed somatostatin (SST) neurotransmitter.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA extracted using total RNA purification micro kit (Norgen cat.35300, Canada). the lysate was treated on column with DNase I to remove DNA contamination. RNA was extracted from the four developmental stage: Day 0 (hESC), Day 26, Day 39, Day 55 in replicates and libraries were prepared by illumine kits.
Illumina Stranded mRNA Prep
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Fastq files were mapped to the hg19 reference using bowtie
Bigwig coverage tracks were generated using deepTools bamCoverage [version 3.4.1]
Coverage peaks were called using MACS [version 2.1.2]
Tommy, explain how the FPKM file was created?
Assembly: hg19
Supplementary files format and content: coverage bigwig file for each sample. A tab-delimited text file with FPKM values for all sample
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| Submission date |
Jan 10, 2023 |
| Last update date |
Mar 15, 2023 |
| Contact name |
Tommy Kaplan |
| E-mail(s) |
tommy@cs.huji.ac.il
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| Organization name |
Hebrew University
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| Department |
School of Computer Science and Engineering
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| Street address |
Givat Ram Campus
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| City |
Jerusalem |
| ZIP/Postal code |
91904 |
| Country |
Israel |
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| Platform ID |
GPL18573 |
| Series (1) |
| GSE218668 |
Epigenetic marks elucidate gene regulatory elements require for human inhibitory interneuron-like progenitors |
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| Relations |
| BioSample |
SAMN32665487 |
| SRA |
SRX18993010 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6926979_day39_rep2.bigwig |
163.3 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Processed data are available on Series record |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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