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| Status |
Public on Dec 15, 2010 |
| Title |
NEC_RNAseq |
| Sample type |
SRA |
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| Source name |
Neuroectodermal sphere cells (NEC) derived from H9 ESC
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| Organism |
Homo sapiens |
| Characteristics |
cell line: NEC
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| Treatment protocol |
hESCs were differentiated into hNECs using a previously described differentiation protocol 21. Briefly, hESC were incubated with 2mg/ml collagenase. Once detached, cells were plated in NEC differentiation media: 1:1 Neurobasal medium/D-MEM F-12 medium (Invitrogen), 0.5x B-27 supplement minus Vitamin A (50x stock, Invitrogen), 0.5x N-2 supplement (100x stock, Invitrogen), 20 ng/ml bFGF (Peprotech), 20 ng/ml EGF (Sigma-Aldrich), 5 µg/ml bovine insulin (Sigma-Aldrich), 0.1 µg/ml recombinant human NOGGIN (Peprotech), 1x Glutamax-I supplement (100x stock, Invitrogen). Cells were differentiated for seven days, changing media every other day.
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| Growth protocol |
hESCs (H9 line, Wi-Cell) were expanded in feeder free, serum free medium, mTESR-1 from StemCell technologies. Cells were passaged 1:7 every 5-6 days by incubation with accutase (Invitrogen) long and resultant small cell clusters were subsequently re-plated on tissue culture dishes coated overnight with growth factor reduced matrigel (BD biosciences). hESCs quality was regularly tested by evaluating the expression of a panel of hESC markers (e.g. Alkaline phosphatase, OCT4) and the capacity to differentiate into cell types derived from the three germ layers.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNAs from hESC and NEC were extracted with Trizol (invitrogen), following manufacturer’s recommendations. 10 µg of total RNA were subjected to two rounds of oligo-dT purification using Dynal oligo (dT) beads (Invitrogen). 100 ng of the purified RNA were fragmented with 10x Fragmentation Buffer (Ambion). Fragmented RNA was used for first strand cDNA synthesis, using random hexamer primers (Invitrogen) and SuperScript II enzyme (Invitrogen). Second strand cDNA was obtained by adding RNAseH (Invitrogen) and DNA Pol I (New England Biolabs) to the first strand cDNA mix. The resulting double-stranded cDNA was used for Illumina library preparation according to Illumina's instructions accompanying the DNA Sample Kit (Part# 0801-0303). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation, DNA was size selected on agarose gel from 200-700 bp. Then isolated DNA was PCR amplified with Illumina primers for 18 cycles. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer |
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| Description |
polyA RNA
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| Data processing |
RNA-seq libraries were sequenced with Illumina Genome Analyzer and both mapping and analysis of resulting reads were perfomed with DNAnexus software tools (https://dnanexus.com)
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| Submission date |
Sep 29, 2010 |
| Last update date |
May 15, 2019 |
| Contact name |
Alvaro Rada |
| E-mail(s) |
alvaror@stanford.edu
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| Organization name |
Stanford University
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| Department |
Chemical and Systems Biology
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| Lab |
Wysocka lab
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| Street address |
269 Campus Drive, CCSR Building
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| City |
Stanford |
| State/province |
CA |
| ZIP/Postal code |
94305 |
| Country |
USA |
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| Platform ID |
GPL9052 |
| Series (1) |
| GSE24447 |
A unique chromatin signature uncovers early developmental enhancers in humans |
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| Relations |
| SRA |
SRX027481 |
| BioSample |
SAMN00113789 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM602290_NEC_RPKM.txt.gz |
518.4 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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