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Sample GSM2467557

Query DataSets for GSM2467557

Status

Public on Jan 30, 2017

Title

PHS_Enhancer_capture_rep2

Sample type

SRA

Source name

Erythrocytes

Organism

Mus musculus

Characteristics

cell type: Erythrocytes
strain: C57BL/6
day of differentiation: Day 5
genes analysed: Hba-1,Hba-2,Hbb-b1,Hbb-b2, Slc25a37,PSE_a,PSE_b,VPE

Treatment protocol

ES cells (ESC) were seeded in suspension at low density (1x104 cells/ml) in the absence of LIF to form embryoid bodies (EBs). ES cells were seeded in DMEM (Invitrogen) supplemented with 15% fetal calf serum (Gibco), 1% Penicillin/Streptomycin (Invitrogen), 0.1mM 2-mercaptoethanol (Sigma), 1% Glutamine (Invitrogen), 1% MEM Non-essential amino acids (Gibco) and 1mM Sodium Pyruvate (Sigma). On day 2 EBs were transferred into N2B27 medium (Cellartis) supplemented with 20ng/ml ActivinA (R&D systems) and 20ng/ml EGF (Peprotech) to induce DE differentiation (Morrison et al. 2008). Single cell suspensions of erythrocytes were made from the spleens of phenylhydrazine treated mice (40mg/g body weight, x3 doses 12h apart, sacrificed on day 5). Erythrocytes were isolated from spleens by gentle manual dissociation, resuspension in PBS and passage through a CellTrics 30 µM filter (Sysmex).

Growth protocol

ES cell lines were maintained in DMEM (Invitrogen) supplemented with 15% fetal calf serum (Gibco), 1% Penicillin/Streptomycin (Invitrogen), 0.1mM 2-mercaptoethanol (Sigma), 1% Glutamine (Invitrogen), 1% MEM Non-essential amino acids (Gibco), 1mM Sodium Pyruvate (Sigma), 1000U/ml LIF (ESGRO) on gelatin coated plates.

Extracted molecule

genomic DNA

Extraction protocol

3C Libraries were generated according to the NG Capture-C protocol (Davies et al. 2016). 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 70%. DNA content of the Dpn II 3C libraries were quantitated using a Qubit fluorometer (Life technologies)
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 Illumina Nextseq 500 (150 bp paired-end reads)

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

Illumina NextSeq 500

Description

DpnII digested 3C library, enriched for fragments of interest by oligonucleotide pulldown
PHS_Enh_Interacting.txt
PHS_B

Data processing

Library strategy: Capture-C
Library Strategy: NG Capture-C
Adaptor Removal: Trim Galore
Fragment reconstruction into a single read: Flash
In silico restriction enzyme digestion of FASTQ file (dpnIIPE.pl)
Alignment: Bowtie 1 maintaining strict read order
Processing: Removal of PCR duplicates; Parsing of informative reads and mapping to restriction enzyme fragments (CCanalyser2.pl)
Processing: Combining data from multiple replicates(custom scripts)
Processing: Removal of ploidy regions and off target capture (custom scripts)
Processing: 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 with count of interactions per fragment (restriction enzyme fragment \t sample 1 \t sample 2 \t etc.)

Submission date

Jan 25, 2017

Last update date

May 15, 2019

Contact name

Damien Downes

E-mail(s)

[email protected]

Phone

01865222374

Organization name

The University of Oxford