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| Status |
Public on Jul 06, 2015 |
| Title |
HiCap_replicate1_mESC |
| Sample type |
SRA |
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| Source name |
Embryonic stem cells
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| Organism |
Mus musculus |
| Characteristics |
cell line: R1
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| Growth protocol |
Mouse embryonic stem cells (line R1) were obtained from Janet Rossant’s lab (Toronto, Canada). Cells were maintained on 0.1% gelatin-coated dishes in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal calf serum (FCS), 0.1 mM non- essential amino acids, 0.3 mg/ml L-glutamine, 1 mM pyruvate (Invitrogen), 1000 U/ml murine LIF (Chemicon International ESGRO), and were kept in a 5% CO2 atmosphere at 37 °C. The medium of undifferentiated cells was changed daily.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were cross-linked with 1% formaldehyde for 10 minutes, lysed and nuclei were isolated. Isolated nuclei were digested with 4-cutter FastDigest MboI (Thermo Scientific, 1µl/µg DNA) for 4 hours at 37 °C. The ends of digested material were filled with biotinylated dATP, dGTP, dCTP and dTTP using Klenow fragment (Fermentas, 0.1 U per 1 µg DNA). Klenow was deactivated using 0.01 M EDTA at incubating 75 °C for 15 minutes. Then the material was diluted to 3.5 ng/µl and ligated using T4 DNA Ligase (Promega). The crosslinking was reversed by adding Proteinase K and incubating overnight at 65 °C. The proteins were removed and DNA was purified using phenol-chloroform followed by ethanol precipitation. Biotinylated but unligated ends were removed using T4 DNA polymerase by incubating at 12 °C for 15 minutes. The material was fragmented to 300-600 bases by sonication. The fragment ends were repaired and A-tailed. Then the biotinylated fragments were bound to streptavidin beads and unbound fragments were washed away. Sequencing adapters were then ligated to the fragments bound to beads. The material was amplified 6-9 cycles while bound to beads to obtain sufficient amount for sequence capture. Original biotinylated material was removed, supernatant was hybridized to sequence capture probe set according to manufacturer’s instructions (Roche Nimblegen Inc.). Hybridized material was washed according to manufacturer’s instructions and amplified with PCR for 3-6 cycles. Hybridization of the probes to the Hi-C material was done exactly according to the manufacturer’s instructions (Roche Nimblegen Inc).
Libraries were prepared for sequencing using Illumina TruSeq protocol
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
HiCap_rep1_promoter-enhancer.gff
Hi-C coupled with sequence capture, biological replicate 1
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| Data processing |
Mapping of reads. Paired-end sequences were aligned to the mouse genome (build mm9) through the tool HiCUP (Babraham Bioinformatics). We then used custom scripts to pair the independently mapped sequence ends and we indexed each sequence pair to their corresponding MboI restriction fragment.
Calling of interactions. We called significant interactions for all promoter containing restriction fragments. To this end, aligned pairs of which at least one mate mapping on a promoter were selected. Promoter regions were defined as 1000 bases downstream and 3000 bases upstream of transcription start site. The extension allowed mappings involving the directly surrounding fragment of the promoter-anchored fragment to be incorporated into analyses, as restriction cut efficiency was only 71%. Nonetheless, in 77% of interactions detected in first HiCap replicate and 92% of interactions detected in the second HiCap replicate, all reads mapped to sequence capture probes. We collected all paired sequences with one end originating from a promoter region and the other end at least 1000 bp away from the promoter region. Next we counted the occurrence of interactions to all MboI restriction fragments in the genome from each captured promoter region. Read pairs with the exact same mapping positions were discarded (to remove any potential effect from PCR duplicates). The same procedure was applied to the negative control regions to obtain read pairs for interactions that were later used as background interaction probabilities. We binned all negative control interactions distances (bin size of 1kb) and calculated the average and standard deviation of the number of interactions found per fragment for negative control regions (discarding fragments with zero interactions from the calculations). These background probabilities of interactions were then used to assess whether each promoter-anchored interaction was significant in each biological replicate independently, using a Z-test. We adjusted the P-values, to account for the multiple tests performed, using the Benjamini-Hochberg procedure and we required a significant interaction to have adjusted P-values below 0.2 in both biological replicates, resulting in an effective adjusted P-value threshold of 0.04 since interactions were required to be present in both biological replicates. Additionally, we required at least 4 supporting read pairs in each biological replicate. Promoter-promoter interactions were called similarly, but requiring that both ends of the paired reads aligned within the annotated promoter regions.
Using significant promoter-enhancer interactions (read threshold 3) we generated a GFF files (for biological replicates separately and for replicated interactions between them) for visualization in Genome Browser.
Genome_build: mm9
Supplementary_files_format_and_content: GFF
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| Submission date |
Aug 18, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Ilgar Abdullayev |
| E-mail(s) |
ilgar.abdullayev@licr.ki.se
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| Organization name |
Karolinska Institutet
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| Department |
Cell and Molecular Biology
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| Lab |
Rickard Sandberg's group (rickard.sandberg@ki.se)
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| Street address |
Nobels väg 3
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| City |
Stockholm |
| ZIP/Postal code |
17177 |
| Country |
Sweden |
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| Platform ID |
GPL13112 |
| Series (2) |
| GSE60494 |
Genome-wide mapping of promoter-enhancer interactions with HiCap [HiCap] |
| GSE60495 |
Genome-wide mapping of promoter-enhancer interactions with HiCap |
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| Relations |
| BioSample |
SAMN02997403 |
| SRA |
SRX682807 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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