GSM5819324: B6xMOLF-H3K27ac-0m-B1; Mus musculus; ChIP-Seq
Media was aspirated from MEFs and 2 mL or 15 mL crosslinking buffer (10 mM HEPES pH 7.5, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA) with 1% formaldehyde was added for 6-well or 15 cm dishes respectively. Cells were crosslinked by shaking gently for 10 min at room temperature. Crosslinking was quenched by adding glycine to a final concentration of 125mM and incubating for 5 min at room temperature while shaking. Cells were washed once in 2 mL or 15 mL PBS for 6-well or 15 cm dishes respectively. Cells were scraped and collected in 1 mL or 5 mL cold PBS for 6-well or 15 cm dishes respectively and pelleted by spinning at 1,000 g for 5 min at 4oC. Crosslinked MEFs per protocol above from 15 cm dishes were resuspended in 1 mL L1 buffer (50 mM HEPES pH 7.5, 140 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.25% Triton X-100, 0.5% NP-40, 10% glycerol, 10 mM sodium butyrate) per 15 cm dish starting material and rotated for 10 min at 4oC to lyse cells. Nuclei were pelleted by spinning at 1,350 g for 5 min at 4oC and resuspended in 1 mL L2 buffer (10 mM Tris-HCl pH 8.0, 200 mM NaCl, 10 mM sodium butyrate) per 15 cm dish starting material and rotated for 10 min at room temperature. Nuclei were pelleted by spinning at 1,350 g for 5 min at 4oC and resuspended in 300 uL LB3 buffer (10 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% sodium deoxycholate, 0.5% N-lauroylsarcosine, 10 mM sodium butyrate) per 15 cm dish starting material. Chromatin was sonicated with a Bioruptor Plus (Diagenode (Denville, NJ, USA)) on “high” power setting with an “on” interval of 30 sec and “off” interval of 45 sec for 36 cycles). DNA concentration was determined by taking 100 uL aliquot of sonicated chromatin, decrosslinking at 95oC for 15 min, and purifying with QIAquick PCR Purification Kit (QIAGEN 28104) and quantifying by Nanodrop. 1 ug of purified chromatin in 10% glycerol was run on 2% agarose gel and stained with ethidium bromide for 30 min to validate fragment size (typically within ~200-1,000 bp). The remainder of the sonicated chromatin was transferred to 1.5 mL tubes and centrifuged at 16,000 g for 10 min at 4oC to pellet insoluble debris. Triton X-100 was added to soluble chromatin to a final 1% concentration. Protein A Dynabeads (Thermo Fisher 10008D) were washed twice in 1 mL cold block solution (0.5% BSA (w/v), 1% Triton X-100, diluted in LB3 buffer). For coupling antibodies to beads, 15 uL bead slurry per IP were resuspended in 1.5 mL cold block solution and the appropriate amount of antibody (0.5 ug for anti-H3K27ac (Abcam (Waltham, MA, USA) ab4729), 0.5 ug for anti-H3K4me1 (Abcam ab8895), 0.5 ug for anti-H3K4me2 (Abcam ab7766), 0.5ug for anti-H3K4me3 (Abcam ab8580), 2 ug for anti-Fos (in-house generated antibody and Santa Cruz Biotechnology (Dallas, TX, USA) sc-7202X), 2 ug for anti-Tead1 (Abcam ab133533), 2 ug for anti-CTCF (Active Motif (Carlsbad, CA, USA) 61312), and 2 ug for anti-JunD (Santa Cruz Biotechnology sc74)) was added before rotating beads for >2 hrs at 4oC. For pre-clearing chromatin, 15 uL bead slurry was added to appropriate amount of chromatin (40 ug for histone modifications, 80 ug for transcription factors) and additional cold LB3 buffer with 1% Triton X-100 was added such that all samples had a final volume of 1.5 mL before rotating samples for > 2 hrs at 4oC. Pre-cleared chromatin was added to antibody-coupled beads and additional cold LB3 buffer with 1% Triton X-100 was added such that all samples had a final volume of 1.8 mL before rotating samples overnight at 4oC. 50 uL of pre-cleared chromatin was stored at -20oC for making input libraries. For all wash steps listed below, samples were rinsed with 1 mL cold wash buffer and rotated for 5 min at 4oC before separating beads with magnet and discarding supernatant. Samples were washed sequentially twice in low salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl), twice in high salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 500 mM NaCl), twice in LiCl buffer (250 mM LiCl, 1% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.0), and once in TE buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA). Samples were eluted from beads by addition of 200 uL TE buffer with 1% SDS and incubating at 65oC for 30 min with brief vortexing every 10 min to mix. IP samples were placed on magnet and supernatant was transferred to new tubes. Input samples were also thawed and 150 uL TE buffer with 1% SDS was added. Both IP and input samples were decrosslinked by incubating at 65oC overnight. 10 ug RNase A (Sigma Aldrich (St. Louis, MO, USA) R6513) was added and samples were incubated at 37oC for 1 hr to digest RNA. 7 uL 20 ug/uL proteinase K (New England Biolabs P8107) was added and samples were incubated at 55oC for 2 hr to digest protein. DNA was extracted with 1 volume of 25:24:1 phenol-chloroform-isoamyl alcohol and purified with QIAquick PCR Purification Kit (QIAGEN 28104). Libraries were prepared with the Ovation Ultralow V2 DNA-Seq Library Preparation Kit (NuGEN (Redwood City, CA, USA) 0344NB-32) per manufacturer's instructions. Libraries were sequenced on an Illumina NextSeq 500 with 150 bp paired-end reads.