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| Status |
Public on Feb 02, 2016 |
| Title |
hESC SOX2/3 KD1 |
| Sample type |
SRA |
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| Source name |
H9 ESC
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| Organism |
Homo sapiens |
| Characteristics |
transfection: first SOX2 and SOX3 siRNAs
time point: 48 hours
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| Treatment protocol |
we used one non-targeting (NT) siRNA, two sets of siRNAs targeting SOX2 and/or SOX3 to KD SOX2 and SOX3 (SOX2/3 KD-1 and SOX2/3-2) in hESCs and KD SOX2 (SOX2 KD-1 and SOX2 KD-2) in hNPCs repectively. hESC and hNPC cells were collected 48 hrs post transfection with siRNAs.
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| Growth protocol |
H9 cells were maintained in mTeSR1 in BD hESC qualified matrigel coated dishes. Culture was daily changed, and hESCs were passaged using Accutase or 1mg/ml dispase. hNPCs were differentiated from H9 cells using established protocol (Chambers SM, Nat Biotechnol, 2009; 27: 275–280). Briefly, we used Noggin (500ng/ml), SB431542 (10mM) and Compound C (10mM) to induce neural differentiaiton.Neural rosettes were picked up and replated onto matrigel coated dishes, and then dissociated into single NPCs and maintained in N2B27 medium supplemented with 10ng/ml bFGF. hNPCs were then passaged using 0.05% trypsin.
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| Extracted molecule |
total RNA |
| Extraction protocol |
collecting the cells in Trizol Reagent ( Invitrogen, 15596026)
The cDNA libraries were constructed following the TruSeq™ RNA Sample Preparation Guide (Illumina). Briefly, total RNA was isolated with Trizol reagent, and the polyA RNA was isolated using the RNA Purification Beads (Illumina). The mRNAs were fragmented by incubation in Elute, Prime, Fragment Mix at 94℃ for 8min to obtain 120-200bp inserts. Fist strand cDNA was synthesized with SuperScript II Reverse Transcriptase (Invitrogen) using random primer, and Ampure XP beads are used to isolated the double‐stranded (ds) cDNA synthesized by Second Strand Master Mix. The adapter was ligated to the A‐Tailing fragment, and 12 cycles of PCR was performed to enrich those DNA fragments that have adapter molecules on both ends and to amplify the amount of DNA in the library. Purified libraries were quantified by Qubit® 2.0 Fluorometer and validated by Agilent 2100 bioanalyzer to confirm the insert size and calculate the mole concentration. Cluster was generated by cBot with the library diluted to 10 pM and then were sequenced on the Illumina Genome Analyzer IIx for 75 cycles. The library construction and sequencing was performed at Shanghai Biotechnology Corporation.
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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 |
Basecalls performed using CASAVA.
RNA-Seq reads of each sample were firstly mapped to the hg19 genome assembly using TopHat 2.0.12 with default parameters.
Read counts were obtained from TopHat-generated BAM files using SAMtools 0.1.16.
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text files include RefSeq id and RPKM values for each Sample
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| Submission date |
Jun 02, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Ying Jin |
| E-mail(s) |
yjin@sibs.ac.cn
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| Organization name |
Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences
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| Street address |
225 South Chongqing Road
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| City |
Shanghai |
| ZIP/Postal code |
200025 |
| Country |
China |
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| Platform ID |
GPL16791 |
| Series (1) |
| GSE69476 |
mRNA sequencing of the global effect of SOX2 on gene expression in hESC and hESC derived NPCs. |
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| Relations |
| BioSample |
SAMN03759633 |
| SRA |
SRX1047408 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1701811_ESC_SOX2_KD_1.txt.gz |
229.8 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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