GSM4421307: P5424_WT_unstim_4C_Ikzf1; Mus musculus; OTHER
Total 40 x106 of wt and ∆IkE120P5424 cells were crosslinked in 1% formaldehyde for 10 min at 20 °C, followed by quenching with glycine at a final concentration of 250 mM. Pelleted cells were washed twice with ice-cold PBS, and then re-suspended in lysis buffer (20 mM Hepes PH 7.6, 1% SDS, 1X PIC) at final cell concentration of 15 x 106 cells/ml. Chromatin was sonicated with Bioruptor (Diagenode) to an average length of 200-400 bp (5 pulses of 30 sec ON and 30 sec OFF). An aliquot of sonicated cell lysate equivalent to 0.5 x 106 cells was diluted with SDS-free dilution buffer (1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris PH 8.0, 167 mM NACL) for single immunoprecipitation. Specific antibodies (1 µg per ChIP) and proteinase inhibitor cocktail were added to the lysate and rotated overnight at 4 °C. The antibody used was as follow H3K27ac (C15410196, Diagenode). On the next day, Protein A magnetic beads (Invitrogen) were washed twice with dilution buffer (0.15% SDS, 1% Triton X-100,1.2 mM EDTA, 16.7 mM Tris pH 8.0, 167 mM NaCl and 0.1% BSA) and added to the lysate and rotated 1 hour at 4 °C. Then, beads were washed with each of the following buffers: once with Wash Buffer 1 (2 mM EDTA, 20 mM Tris pH 8.0, 1% Triton X-100, 01% SDS, 150 mM NaCl), twice with Wash Buffer 2 (2 mM EDTA, 20 mM Tris pH 8.0, 1% Triton X-100, 0.1% SDS, 500 mM NaCl), twice with Wash Buffer 3 (1 mM EDTA, 10 mM Tris pH 8.0). Finally, beads were eluted in Elution buffer (1% SDS, 0.1 M NaHCo3) and rotated at RT for 20 min. Eluted materials were then added with 0.2 M NaCl, 0.1 mg/ml of proteinase K and incubated overnight at 65 °C reverse cross-linking, along with the untreated input (10% of the starting material). The next day, DNA was purified with QIAquick PCR Purification Kit (Qiagen) and eluted in 30 µl of water. For 4C, cells were detached with trypsin (for ES cells), washed by centrifugation in PBS, and then fixed with 2% formaldehyde in mES culture medium for 10 min at 23°C. The fixation was quenched with cold glycine at a final concentration of 125 mM, then cells were washed with PBS and permeabilized on ice for 1 h with 10 mM Tris-HCl, pH 8, 100 mM NaCl, 0.1% NP-40 and protease inhibitors. Nuclei were resuspended in DpnII restriction buffer at 10 million nuclei/mL concentration, and 5 million nuclei aliquots were further permeabilized by treatment for 1 h with 0.4% SDS at 37°C, then a further 1h with 2.6% Triton-X100 at 37°C. Nuclei were digested overnight with 1000 U DpnII at 37°C, then washed twice by centrifuging and resuspending in T4 DNA ligase buffer. In situ ligation was performed in 400 μL T4 DNA ligase buffer with 20,000 U T4 DNA ligase overnight at 16°C. DNA was purified by reverse cross-linking with an overnight incubation at 65°C with proteinase K, followed by RNase A digestion, phenol/chloroform extraction and isopropanol precipitation. The DNA was digested with 5 U/μg Csp6I at 37°C overnight, then re-purified by phenol/chloroform extraction and isopropanol precipitation. The DNA was then circularized by ligation with 200 U/μg T4 DNA ligase under dilute conditions (5 ng/μL DNA), and purified by phenol/chloroform extraction and isopropanol precipitation. 50 ng aliquots of this DNA were used as template for PCR with bait-specific primers containing Illumina adapter termini (primer sequences and optimal PCR conditions available on request) At least 1ng of ChIP was used for library preparation. Libraries for ChIPs against H3K27ac was prepared according to Illumina ChIP-Seq protocol and sequenced on a Nextseq500 (Illumina) according to the manufacturer's instructions. For 4C, PCR reactions were pooled, primers removed by washing with 1.8x AMPure XP beads, then quantified on a Bioanalyzer (Agilent) before sequencing with a HiSeq 4000 (Illumina). Paired-end sequencing 50+30bp was performed using Illumina NextSeq 500. For 4C, single-end sequencing (50 nt) on Illumina HiSeq 4000.
GEO Accession
GSM4421307