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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Sep 22, 2021 |
| Title |
D3839 EBd7 CD71+ Rhbdf1 and Mpg Captures sample 2 (CTCFDKO_A81_5Promoters) |
| Sample type |
SRA |
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| Source name |
Chromatin from purified CD71+ erythroid cells derived from differentiated D3839 mES cells, Rhbdf1 and Mpg promoters as Capture point, sample 2
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| Organism |
Mus musculus |
| Characteristics |
cell type: E14TG2a.IV mES derived Erythroid cells
strain: 129/Ola
analysis: Alpha Globin Locus
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
To produce CD71+ erythroid cells in vitro from differentiated mESCs (WT or mutant D3839 which is a mouse ESC line where mouse alpha globon HS38 and HS39.5 indicating CTCF sites/5' boundary elements have been deleted): 24h prior to differentiation, mESCs were induced by passaging into IMDM base media, trypsinised and plated in differentiation media (IMDM suplemented serum and other reagents and growth factors) in either triple vent petri dishes (Thermo Fisher) or flat-bottom 96-well plates (Thermo Fisher) at 3x104 cells in 10 cm dishes or 250 cells per well of a 96-well plate for up to seven days without disruption. EBs were harvested and disaggregated in 0.25% trypsin for 3 minutes at 37°C. CD71-positive cells were isolated by magnetic column separation (LS Column, Miltenyi), according to the manufacturer’s instructions. Briefly, cells (from disaggregated EBs) were labelled with anti-mouse CD71-FITC (eBioscience 11-0711-85; 1:200) in staining buffer (PBS with 10% FCS; 500 ul per 10^7 cells) for 20 minutes at 4°C, washed, then incubated with MACS anti-FITC separation microbeads (Miltenyi; 10 ul per 10^7 cells, following the manufacturer’s instructions) and bead-labelled cells were retained by LS columns. Cells were then processed for Capture-C as in Davies J et al, Nature Methods.
The protocol used for generation of the 3C libraries was similar to previous methods with minor modifications. Briefly, cells were crosslinked with 2% formaldehyde (10 minutes, room temperature); quenched with cold glycine; washed in phosphate buffered saline; resuspended in cold lysis buffer (tris 10mM, NaCl 10mM, NP40 0.2%, complete proteinase inhibitor (Roche)) and snap frozen to -80. Cells were thawed on ice, washed in milliq dH2O and Dounce homogenised on ice (x 40 strokes). Cells were then resuspended with 0.25% SDS and restriction enzyme buffer and incubated at 37C for 1h at 1400rpm on a Comfort Thermomixer (Eppendorf) followed by a further incubation of 1h following the addition of triton X100 (final concentration 1.67%). An overnight digestion was performed using Dpn II (500U /ml (NEB) at 37C / 1400 rpm). The digested chromatin was ligated overnight (Fermentas HC Ligase final concentration 10U/ml) at 16 degrees at 1400 rpm on the Thermomixer. The samples were then decrosslinked overnight at 65C with Proteinase K (Roche) followed by a 30 min incubation at 37C with RNAse (Roche). Phenol/Chloroform extraction was then performed followed by an Ethanol precipitation and a wash with 70% Ethanol. Digestion efficiencys were assessed by gel electrophoresis (1% agarose) and RT-PCR (Taqman), which showed digestion efficiencies in excess of 80%. DNA content of the Dpn II 3C libraries were quantitated using a Qubit fluorometer (Life technologies) and 5-10ug of each library was sheared using a Covaris S2 in milliq dH2O. Covaris settings used were: duty cycle 10%, Intensity 5, Cycles/burst 200, Time 6 cycles of 60seconds, Set Mode Frequency sweeping Temperature 4 to 7 degrees. Following shearing DNA was purified using AMPureXP beads (Agencourt) and DNA quality assessed on a Bioanalyser 2100 using a DNA High Sensitivity Chip (Agilent). DNA end repair and adapter ligation was performed using the NEB Next or NEB Ultra II DNA sample preparation reagent kits, depending on the amount of DNA available, using the standard protocol. Biotinylated capture oligonucleotides were designed to the ends of the viewpoint fragments. Where possible 1-2ug of each adapter ligated library were hybridized with the biotinylated capture oligonucleotides, using the Nimblegen SeqCap reagents and an adapted protocol. The quality of the resultant captured library was assessed by Agilent tapestation or bioanalyser (D1000). The resulting libraries were sequenced using Illumina Next-Seq.
The experimental strategy was to generate very high complexity randomly sonicated 3C libraries with ligated Illumina sequencing adaptors (ideally with several hundred thousand fold coverage). Oligonucleotide capture technology was then used to pull down the restricion fragments containing the viewpoints of interest and their interacting partners, which were subsequently idendified by high throughput sequencing. The power of the technique is that multiple samples and genes can be analysed simultaneously by using differently indexed samples.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina MiSeq |
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| Description |
sequences enriched from a 3C library for their interaction with the Capture point in the Alpha Globin locus as detailed in the sample name
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| Data processing |
Library strategy: Capture-C-seq
Trim_galore (to remove sequencing adaptors)
FLASH (to reconstruct paired end reads into single reads where possible)
In silico restriction enzyme digestion of FASTQ file (dpnIIPE.pl)
Alignment to the genome (Bowtie) maintaining strict read order
Removal of PCR duplicates; Parsing of informative reads and mapping to restriction enzyme fragments (CCanalyser2.pl)
Combining data from multiple replicates (custom scripts)
Removal of ploidy regions and off target capture (custom scripts)
Analysis in R to normalise between tracks using the total number of informative interactions
Genome_build: mm9
Supplementary_files_format_and_content: Custom combined data format (restriction enzyme fragment \t sample 1 \t sample 2 \t etc.)
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| Submission date |
Sep 20, 2021 |
| Last update date |
Sep 23, 2021 |
| Contact name |
Mira Tony Kassouf |
| E-mail(s) |
mira.kassouf@imm.ox.ac.uk
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| Organization name |
University of Oxford
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| Department |
Weatherall Institute of Molecular Medicine
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| Lab |
Doug Higgs
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| Street address |
John Radcliffe Hospital, Headly way
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| City |
Oxford |
| State/province |
England |
| ZIP/Postal code |
OX3 9DS |
| Country |
United Kingdom |
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| Platform ID |
GPL16417 |
| Series (2) |
| GSE184432 |
Scalable Production of Mouse Erythroid Cells using an in vitro Embryoid Body differentiation System [Capture-C] |
| GSE184435 |
Scalable Production of Mouse Erythroid Cells using an in vitro Embryoid Body differentiation System |
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| Relations |
| BioSample |
SAMN21516527 |
| SRA |
SRX12260402 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5588960_CTCFDKO_A81_5Promoters_interaction_counts.tab.gz |
929.8 Kb |
(ftp)(http) |
TAB |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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