GSM1156608
GSM1156608: Ter119_2_Capture-C; Mus musculus; OTHER
The protocol used for generation of the 3C libraries was similar to previous methods with minor modifications. Briefly, 107 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 37oC 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 37 degrees / 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 65 oC with Proteinase K (Roche) followed by a 30 min incubation at 37 oC 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 5ug 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 DNA sample preparation reagent kit using the standard protocol. Excess adapters were removed using AMpureXP beads. 500ng of each adapter ligated library were used as input for the SureSelect target enrichment kit using the recommended protocol and the custom designed Capture-C library. The quality of the resultant captured library was assessed by Bioanalyser using the high sensitivity Chip. The generated libraries fell within the expected guidelines for output of the SureSelect target enrichment kit and were submitted for Hi-Seq paired sequencing (50 bp read length).
GEO
GSM1156608
GEO Accession
GSM1156608