alias: GSM2318516
title: GSM2318516: P2cc control; Mus musculus; OTHER
library_construction_protocol: Cells were cross-linked in 2% formaldehyde, 5% FBS and 1x PBS for 10 minutes at room temperature while rotating. Formaldehyde was quenched with a final concentration of 125 mM glycine for 5 minutes on ice while inverting several times. Cell pellets were washed twice with ice-cold 1x PBS.   Nuclei were harvested by lysing the cell pellets in ice-cold lysis buffer (10 mM Tris pH 8.0, 10 mM NaCl, 0.2% NP-40, protease inhibitors) for 10 minutes on ice. Nuclei were then resuspended in 1.2x DpnII restriction buffer (New England Biolabs) and 0.3% SDS and incubated for 1 hour at 37°C while shaking. Triton X-100 was then added to a final concentration of 1.8% and the reaction was left at 37°C while shaking for another hour. Chromatin was digested with 800 U of DpnII overnight at 37°C while shaking. DpnII was inactivated by adding SDS to a final concentration of 1.3% and incubating at 65°C for 20 minutes. Nuclei were diluted into a volume of 7 mL containing 1.01x T4 DNA ligase buffer (Life Technologies) and Triton-X100 at a final concentration of 1% and incubated at 37°C for 1 hour. Ligations were carried out with 100 U of T4 ligase (Life Technologies) for 4.5 hours at 16°C and 30 minutes at room temperature while shaking. For control libraries, ligase was omitted. Samples were proteinase K treated and reverse-crosslinked overnight at 65 °C. Samples were then treated with RNase A at 37 °C for 30 minutes. DNA was purified by phenol/chloroform extraction and ethanol precipitated.   A second digestion was performed with 25 U of BfaI (New England Biolabs) for P1 and P2 baits or MseI (New England Biolabs) for +24 baits in restriction buffer overnight at 37 °C while shaking. Restriction was inactivated by adding SDS to a final concentration of 1.3% and incubating at 65°C for 20 minutes. A second ligation was performed in the same way as the first ligation, except that ligations were incubated overnight. DNA was purified with two phenol/chloroform extractions and one chloroform extraction followed by ethanol precipitation. DNA concentrations were measured using Qubit® 3.0 Fluorometer (Life Technologies) and Qubit® double-stranded DNA (dsDNA) High Sensitivity Assay kit (Life Technologies).  For each bait, a total of 1 μg of DNA was amplified by PCR using Q5® High-Fidelity DNA Polymerase (New England Biolabs). PCR products were purified using the QIAquick PCR Purification kit (QIAGEN). Three libraries were generated, two replicates and one control (a 1:1 mix of both replicates in which the first ligation step was omitted). Amplicon DNA concentrations for each bait were measured using Qubit® 3.0 Fluorometer and the average fragment sizes by 2100 Bioanalyser (Agilent Technologies) using a High Sensitivity DNA Kit (Agilent Technologies). Amplicons from the six different baits were mixed equally based on the concentration, the average fragment size and the ratio of demultiplexed 4C baits obtained from an initial MiSeq run. Libraries were prepared with the Prep2Seq DNA Library Prep Kit from Illumina (Affymetrix) and TruSeq® barcoded adaptors #2, #7 and #19 from set A (Illumina). Libraries were mixed equimolarly and sequenced as 125 bp paired-end reads on two Illumina HiSeq 2500 lanes.
identifiers.namespace: GEO
identifiers.id: GSM2318516