|
| Status |
Public on Jul 01, 2012 |
| Title |
hESC H9 |
| Sample type |
SRA |
| |
|
| Source name |
embryonic pluripotent stem cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: hESC H9
|
| Treatment protocol |
No specific treatment
|
| Growth protocol |
Human Embryonic Stem Cells H9 (hESC H9) were maintained on matrigel with chemically defined medium as previously described (Ludwig, T.E. et al. Feeder-independent culture of human embryonic stem cells. Nat Methods 3, 637-46 (2006)).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
We purified short nascent strands at replication origins in different cell types. We realized isolation of Short Nascent Strands at replication origins (SNS) of asynchronous human cells by adapting the protocol previously described (Cadoret et al. PNAS 2008). For each cell type, we purified the SNS of two biological replicates. For each sample, DNAzol was used to extract DNA of 100 millions asynchronous cells. Then DNA fragments of 1-2 kb were isolated after sucrose gradient separation. After two runs of lambda exonuclease, the quality for each purified SNS preparation was tested, as previously described (Cadoret et al. PNAS 2008). Synthesis of second strands was realized with the kit bioprime DNA labeling system. In order to be reproducible, we realized, for each biological sample, this synthesis with the same amount of background DNA calculated on the basis of background around the replication origin at the Myc locus. In order to be able to compare the replication origins between different cells, we gave the same amount of background DNA to the sequencing platform.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina Genome Analyzer II |
| |
|
| Description |
enrichment of short nascent strands (SNS) at replication origins with lambda exonuclease
|
| Data processing |
The next generation sequencing of purified and sonicated SNS was realized in the Montpellier GenomiX (MGX) facility in Montpellier, France using the Illumina’s sequencing by synthesis technology. The sequencing library was clusterized and then hybridized with sequencing primers. The sequencing of 36 bp reads by single-end pairing was performed with HiSeq 2000 of Illumina.
Image analyses were performed using HiSeq Control Software provided by Illumina.
Basecalling performed using RTA software provided by Illumina.
Sequencing reads were aligned to the hg18 genome assembly using CASAVA software (version 1.7).
Genomic position of unique sequenced reads were obtained.
To analyse the enrichment of short nascent strand with the Sole-Search tool (http://havoc.genomecenter.ucdavis.edu/cgi-bin/chipseq.cgi), we generated bed files from the retrieved list of sequenced reads with 6 columns.
To retrieve the maximum of replication origin positions for each cell type, we analyzed the SNS enrichment on the union set of sequencing data from two biological replicates. We compared the identified replication origins in the different cell types.
Genome_build: hg18
Supplementary_files_format_and_content: bed files with position of uniquely mapped sequenced reads, DNA sequence and strand.
Supplementary_files_format_and_content: hESC_H9.bed We analyzed the SNS enrichment on this union set of sequenced reads for further analyses
Supplementary_files_format_and_content: H9_cluster.bed We analyzed the SNS enrichment on this union set of sequenced reads for cluster analyses
|
| |
|
| Submission date |
May 04, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Emilie Besnard |
| Organization name |
Institute of Functional Genomics
|
| Lab |
Genome Plasticity and Aging
|
| Street address |
141 rue de la Cardonille
|
| City |
MONTPELLIER |
| ZIP/Postal code |
34000 |
| Country |
France |
| |
|
| Platform ID |
GPL9115 |
| Series (2) |
| GSE37757 |
Unraveling cell type–specific and reprogrammable human replication origin signatures associated with G-quadruplex consensus motifs |
| GSE51334 |
DNA replication-timing boundaries separate stable chromosome domains with cell-type-specific functions |
|
| Relations |
| SRA |
SRX145992 |
| BioSample |
SAMN00990946 |
