|
| Status |
Public on Apr 28, 2014 |
| Title |
4C_EScells_CNS(39) |
| Sample type |
SRA |
| |
|
| Source name |
ES cells
|
| Organism |
Mus musculus |
| Characteristics |
strain: E14
tissue: Embryonic stem cells
age: -
genotype: WT
|
| Treatment protocol |
4C–seq libraries were constructed as described before (Noordermeer et al. 2011, Science). ES cell 4C-seq libraries were prepared from samples consisting of 20 million cells. The primary restriction enzyme used was NlaIII (New England Biolabs, R0125L) and the secondary restriction enzyme was DpnII (New England Biolabs, R0543M). For each viewpoint approximately 1 μg of the 4C-seq library was amplified using 16 individual PCR reactions with inverse primers containing Illumina Solexa adapter sequences.
Circular Chromosome Conformation Capture (4C) technique was performed as described (Noordermeer et al, 2011 Science). Cross-linked chromatin was digested with NlaIII, ligated under diluted conditions, cross links were reversed and DNA was further digested by DpnII and again ligated under diluted conditions.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Multiplexed samples were sequenced on the Illumina HiSeq system using 100 bp single-end reads according to the manufacturer’s specifications. 4C-seq reads were sorted, aligned, and translated to restriction fragments using the 4C-seq pipeline of the BBCF HTSstation (David et al. 2014, Plos ONE and available at http://htsstation.epfl.ch). Samples were mapped to the ENSEMBL Mouse assembly NCBIM37 (mm9).
Illumina adapters (single end reads) were included in the inverse 4C PCR primers used for 4C-seq library preparation
Cells were isolated in PBS supplemented with 10% Fetal Calf Serum. Tissue sample were further made single cell by collagenase treatment and applying a cell-strainer cap (BD Biosciences). Chromatin was crosslinked in the presence of 2% formaldehyde.
PCR amplification using primers with included adapter sequences (for Illumina HiSeq2000)
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina Genome Analyzer II |
| |
|
| Data processing |
De-multiplexing, mapping and 4C-seq were done through HTSstation (David et al, 2014 PLoS ONE and http://htsstation.epfl.ch/).
Reads were mapped to the mm9 (NCBIM37) Mouse genome assembly with bowtie version 0.12.7 (Langmead et al, 2009 Genome Biology) with parameters -n 2 --best -strata -m 5.
Mapped reads were translated to restriction fragments using the HTSstation (David et al, 2014 PLoS ONE and http://htsstation.epfl.ch/).
4C figures were made using a running mean algorithm with a window size of eleven fragments.
Genome_build: mm9
Supplementary_files_format_and_content: Signal/restriction fragment (bedGraph) and smoothed (bedGraph), one per viewpoint, per tissue
|
| |
|
| Submission date |
Feb 25, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Marion LELEU |
| Organization name |
EPFL
|
| Department |
School of Life Sciences
|
| Lab |
Laboratory of developmental genomics
|
| Street address |
EPFL-SV-ISREC-UPDUB- Station 19
|
| City |
Lausanne |
| ZIP/Postal code |
1015 |
| Country |
Switzerland |
| |
|
| Platform ID |
GPL9250 |
| Series (2) |
| GSE55342 |
4C-seq: Temporal dynamics and developmental memory of 3D chromatin architecture at Hox gene loci |
| GSE55344 |
Temporal dynamics and developmental memory of 3D chromatin architecture at Hox gene loci |
|
| Relations |
| BioSample |
SAMN02664779 |
| SRA |
SRX475956 |
