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Sample GSM2077402

Query DataSets for GSM2077402

Status

Public on Jun 02, 2016

Title

Fetal liver Knockout of alpha globin regulatory element R3 [Capture C]

Sample type

SRA

Source name

Erythroid cells

Organism

Mus musculus

Characteristics

knockout of enhancer-like component: regulatory element R3
strain: F1 C57Bl6 / CBA
tissue: Fetal Liver Culture
genes analysed: Hba-a1&2, Hbb-b1&2, Slc25A37

Extracted molecule

genomic DNA

Extraction protocol

Fetal livers were dissected from E12.5 mouse embryos and the dissociated cell suspension was expanded for 5-7 days in Stempro (Invitrogen) supplemented with Epo (1U/ml), SCF (50ng/ml) and dexamethasone (1mM). Following expansion of the cultures, mature red cells were removed by negative selection for Ter119 using anti-Ter-119 magnetic beads (Miltenyi), to obtain a population of erythroid precursors. Cultures were then switched to medium containing high Epo (5U/ml) to induce differentiation to mature erythroid cells. After 18hrs differentiation, 3C libraries were prepared.
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 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 either the Illumina MiSeq (150bp paired end reads) or HiSeq (100bp 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.

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

Illumina NextSeq 500

Description

molecule: Next generation Capture-C product
3C library prepartion followed by double oligonucleotide capture
HbaCombined_KO_PF_BF.gfc
HbbCombined_KO_PF_BF.gfc
Slc25A37_KO_PF_BF.gfc

Data processing

Library strategy: Capture-C
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.)

Submission date

Mar 01, 2016

Last update date

May 15, 2019

Contact name

Jim Hughes

E-mail(s)

jim.hughes@imm.ox.ac.uk

Phone

1865222113

Organization name

University of Oxford