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| Status |
Public on Nov 05, 2009 |
| Title |
ES-S-lane3 |
| Sample type |
SRA |
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| Source name |
Human embryonic stem cells
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| Organism |
Homo sapiens |
| Characteristics |
cell type: embryonic stem cells
cell cycle phase: S
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| Growth protocol |
matrigel
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA from sorted cells in S or G1 was extracted, sheared by sonication to a length of about 500bp and Solexa linkers were ligated using standard procedures. Purified DNA is subjected to mechanical fragmentation by nebulization (compressed air at 32-35psi for 6 minutes on ice). The resulting double-stranded (ds) overhang fragments are end-repaired by incubation in the presence of T4 DNA polymerase and Klenow polymerase. The polished fragments are phosphorylated by T4 PNK, followed by the addition of a single ‘A’ base to the 3' end of the blunt-ended phosphorylated fragments. This ‘A’ base prepares the DNA fragments for ligation to proprietary adapter oligonucleotides (Illumina single-read or paired-read sequences) which have a ‘T’ base at their 3' end. Ligation products are size-selected by gel electrophoresis and purification (2% low-range agarose with ethidium bromide). Following 1-2 hours at 80-110V (room temperature), the library range is visualized under brief UV and the desired size (200-500bp) excised with a clean scalpel. Purified DNA libraries are subjected to a final PCR amplification step (15 cycles). All amplified libraries are quantitatively and qualitatively assessed by Nanodrop ND-1000 (Thermo Scientific, DE, USA) UV/Vis spectroscopy and DNA bioanalyzer 2100 (Agilent, CA, USA) microfluidics.
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| Library strategy |
WGS |
| Library source |
genomic |
| Library selection |
PCR |
| Instrument model |
Illumina Genome Analyzer II |
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| Description |
Human ES cells, S phase
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| Data processing |
The data were processed using the GAPipeline-1.3.2, which includes
1. Image analysis (Firecrest)-Uses the raw TIF files to locate clusters on the image, and outputs the cluster intensity, X,Y positions, and an estimate of the noise for each cluster. The output from image analysis provides the input for base calling.
2. Base calling (Bustard)-Uses cluster intensities and noise estimate to output the sequence of bases read from each cluster, along with a confidence level for each base.
3. Sequence analysis (Gerald and Eland)-Allows for alignment to a reference sequence, filtering of data based on predefined criteria, and visualization of the result. Uniquely matching reads (with 0, 1 or 2 mismatches) were further processed with custom smoothing algorithms. See publication (PMID:19767418).
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| Submission date |
Nov 02, 2009 |
| Last update date |
May 15, 2019 |
| Contact name |
Eric Bouhassira |
| E-mail(s) |
[email protected]
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| Organization name |
Albert Einstein College of Medicine
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| Street address |
1300 Morris Park avenue
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| City |
The Bronx |
| ZIP/Postal code |
10461 |
| Country |
USA |
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| Platform ID |
GPL9115 |
| Series (1) |
| GSE18679 |
TimEX from human embryonic stem cells, mesenchymal stem cells, and erythroid cells |
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| Relations |
| SRA |
SRX015983 |
| BioSample |
SAMN00008178 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM467175_s_7_eland_extended.txt.gz |
202.1 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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