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Status |
Public on Jun 14, 2017 |
Title |
Mouse erytrhoid cells from knockout of CTCF binding site -HS38 and -HS39 replicate 2, Enhancer R1 capture |
Sample type |
SRA |
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Source name |
Erythroid cells
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Organism |
Mus musculus |
Characteristics |
strain: C57Bl6 tissue: Phenylhydrazine treated spleen genes analysed: alpha globin enhancer R1
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Treatment protocol |
Ter119+ cells were isolated from mouse spleens
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Growth protocol |
C57BL/6 mice were treated with phenylhydrazine (three doses of 40 mg/g body weight given 12 hours apart; mice were sacrificed after five days)
|
Extracted molecule |
genomic DNA |
Extraction protocol |
A single cell suspension was made by gently dissociating the spleen of a phenyhydrazine treated mouse and passing it through a 70um filter. Ter119 selection was performed by staining the cells with phycoerythrin conjugated anti-ter119 antibodies (BD biosciences). The cells were subsequently washed and then incubated with MACS anti-phycoerythrin beads. Cells were then separated using an auto-MACS (Miltenyi Biotec). NG Capture-C combines 3C library preparation with oligonucleotide capture for the desired viewpoint restriction fragments. Detailed description follows. The protocol used for generation of the 3C libraries: 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 sample preparation reagent kits, 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 the Illumina MiSeq (150bp paired end reads). 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 promoters of the genes 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 |
Next generation Capture-C products : 3C library prepartion followed by double oligonucleotide capture A1_R1_DEL3839vsC57BL6.gfc
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Data processing |
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 -m 2) maintaining strict read order Removal of PCR duplicates; Parsing of informative reads and mapping to restriction enzyme fragments (CCanalyser4.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 |
Apr 17, 2017 |
Last update date |
May 15, 2019 |
Contact name |
Jelena M Telenius |
E-mail(s) |
jelena.telenius@ndcls.ox.ac.uk
|
Organization name |
Oxford University
|
Department |
Weatherall Institute of Molecular Medicine (WIMM)
|
Lab |
Genome Biology Research Group
|
Street address |
MRC Weatherall Institute of Molecular Medicine University of Oxford John Radcliffe Hospital Headington
|
City |
Oxford |
ZIP/Postal code |
OX3 9DS |
Country |
United Kingdom |
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Platform ID |
GPL16417 |
Series (2) |
GSE97867 |
Tissue-specific CTCF/Cohesin-mediated chromatin architecture delimits enhancer interactions and function in vivo (Capture-C" |
GSE97871 |
Tissue-specific CTCF/Cohesin-mediated chromatin architecture delimits enhancer interactions and function in vivo |
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Relations |
BioSample |
SAMN06759668 |
SRA |
SRX2741766 |