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| Status |
Public on Jan 30, 2019 |
| Title |
E14-serum_2 |
| Sample type |
SRA |
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| Source name |
Mouse ES cells
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| Organism |
Mus musculus |
| Characteristics |
culture condition: Serum+LIF
biologial replicate: 2
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| Growth protocol |
Cells were maintained at 37°C with 5% CO2 and passaged every 2-3 days. Serum cells were maintained in either GMEM (Gibco) supplemented with 15% foetal calf serum, 0.1mM nonessential amino acids (SIGMA), 1mM sodium Pyruvate (Sigma) 1% Penicillin/Streptomycin, 2mM L-glutamine, 0.1mM β-mercaptoethanol (Thermo Fisher), and ESGRO LIF (Millipore) at 1000U/mL. Cells were grown on 0.2% gelatin (Sigma). 2i culture conditions include 50% DMEM/F12 (Gibco), 50% Neurobasal media (Gibco), 0.5% N2 supplement, 1% B27 & RA (Gibco), 7.5% BSA (Gibco), 1% Penicilllin/Streptomycin, 2mM L-glutamine, Monothioglycerol 1.5 x 10-4M (Sigma), 1000U/ml ESGRO LIF (Millipore), 1uM PD0325901 (MEK inhibitor, Stemgent) and 3Um CHIR99021 (GSK3 inhibitor, Stemgent). mESCs were passaged every 2-3 days using trypsin/EDTA (Sigma). 2i conversions were carried out for 14 days.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Hi-C was performed largely as described (Rao et. el., 2014) with minor modifications. Briefly, ES cells were crosslinked in 1% formaldehyde for 10 minutes, snap-frozen in 2-5 mln aliquots and stored at -80°C. After permeabilization in lysis buffer (0.2% Igepal, 10 mM Tris-HCl pH 8.0, 10 mM NaCl, 1x Halt Protease inhibitor cocktail) nuclei were isolated in 0.3% SDS in NEBuffer 3 at 62° for 10 minutes. SDS was quenched with 1% Triton X-100 at 37° for 1 h, then the nuclei were pelleted and resuspended in 250 ul DpnII buffer with 600 U DpnII. After overnight digestion, 200 more units were added for 2 hours. Then the ends were filled-in using Klenow, d(G/C/T)TPs and biotin-14-dATP for 1.5 hrs at 37°. After ligation at room temperature for 4 hrs the nuclei were spun down, resuspended in 200 ul mQ and digested with proteinase K for 30 min at 55° in presence of 1% SDS. Then the cross-links were reversed at 65° overnight after addition of NaCl to a final concentration of 1.85 M. In the morning, after ethanol precipitation and a wash with 70-80% Ethanol, DNA was resuspended in 500 ul of sonication buffer (50 mM Tris pH 8.0, 0.1%SDS, 10 mM EDTA), incubated on ice for 15 min and then sheared using a probe sonicator to fragment size of 200-700 bp. Then the DNA was concentrated on Amicon filter units, bound to MyOne T1 Streptavidin beads and used for Illumina library preparation. Small aliquots were taken before and after DpnII treatment, and before sonication to confirm efficient DNA digestion and ligation by running them on 1% agarose gel. Samples were first test-sequenced on NextSeq 550 (WTCRF, Edinburgh) to check library quality, and then selected libraries were sequenced deeply on HiSeq 4000 (BGI).
In house Illumina library protocol from Rao et. al., 2014.
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| Library strategy |
Hi-C |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
Fastq files were generated by the sequencing facilities (Wellcome Trust Clinical Research Facility for test sequencing on NextSeq 550, BGI for deep sequencing on HiSeq 4000).
Reads were processed using distiller (https://github.com/mirnylab/distiller-nf) on the high-performance computing cluster of the University of Edinburgh (Eddie3). Mapping was performed to the mm9 genome build. Hi-C pairs with exactly matching coordinates were removed as PCR or optical duplicates (pcr_dups_max_mismatch_bp: 0). Only read pairs with both mapq>30 were retained. The output statistics information and multiresolution Cooler files were used in downstream analyses.
For pileup analysis, we took all regions of interest in the Hi-C maps, e.g. all cis interactions between CGIs bound or not bound by RING1B, and averaged a 15×15 window centered on them at 5 Kb resolution (10 Kb for the Du et al data, and comparison with it). For each of those windows 10 control windows were obtained by randomly shifting the position of the original window between 100 kb and 1 Mb along the diagonal of the map. All control windows were averaged, and were used to normalise the pile-up to remove the distance-dependency of interactions. Then, the final matrix was divided by the average value of its top-left and bottom-right 3×3 corners, to normalize the local background. Values of enrichment are the enrichment of interactions in the center pixel of the matrix, after all described normalization procedures. The code used to perform this analysis is available here: https://github.com/Phlya/CoolPUp
genome build: mm9
processed data files format and content: We provide 4 processed files for each biological sample, and three for combined data for each condition. They are (i) a gzip-compressed .pairs file (https://pairtools.readthedocs.io/en/latest/index.html, except W are called C, and U are called L), according to an older .pairtools format convention), (ii) 5 kb resolution .cool files (https://github.com/mirnylab/cooler), (iii) 10 kb resolution .cool files, and (iv) multiresolution .cool files storing data in resolution 1*2^n kb, which can be used in HiGlass (https://github.com/hms-dbmi/higlass) for interactive visualization.
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| Submission date |
Dec 24, 2018 |
| Last update date |
May 10, 2019 |
| Contact name |
Ilya M. Flyamer |
| E-mail(s) |
flyamer@gmail.com
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| Organization name |
Friedrich Miescher Institute for Biomedical Research
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| Lab |
Giorgetti
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| Street address |
Maulbeerstrasse 66
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| City |
Basel |
| ZIP/Postal code |
4058 |
| Country |
Switzerland |
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| Platform ID |
GPL21103 |
| Series (1) |
| GSE124342 |
Hi-C of WT E14 mouse ES cells grown in serum and 2i media |
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| Relations |
| BioSample |
SAMN10642588 |
| SRA |
SRX5179251 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3530162_serum-2.mm9.mapq_30.1000.mcool |
1.6 Gb |
(ftp)(http) |
MCOOL |
| GSM3530162_serum-2.mm9.nodups.pairs.gz |
6.5 Gb |
(ftp)(http) |
PAIRS |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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