alias: GSM2875629
title: GSM2875629: Mouse Fetal liver cells, NextSeq, 30 hours, biol rep 2, Captured from: HS39.5, Hba-1, Hba-2; Mus musculus; OTHER
library_construction_protocol: 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 efficiencies 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 were 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 restriction fragments containing the promoters of the genes of interest and their interacting partners, which were subsequently identified by high throughput sequencing. The power of the technique is that multiple samples and genes can be analysed simultaneously by using differently indexed samples.
identifiers.namespace: GEO
identifiers.id: GSM2875629
attributes.tag: GEO Accession
attributes.value: GSM2875629