GSM1523573: FL17_H3K27ac; Homo sapiens; ChIP-Seq
ChIP-Seq: Cells were fixed with 1% formaldehyde in culture medium for 10 min at room temperature followed by quenching with 0.125 M glycine for 5 min. The cells were washed with ice-cold PBS and lysed in SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris pH8.0) for 20 min on ice. The crosslinked chromatin was sheared to an average size of less than 300 bp using a BiorupterTM sonicator (Diagenode). 5 µg of antibody was incubated with Dynabeads ProteinA beads (Invitrogen at 4C for 2-4 hours .  Sonicated lysates were subjected to immunopreciptation overnight at 4°C with acetylated H3 (Millipore, #06-599, Lot#2277850), acetylated H3K27 (Abcam,#ab4739, Lot#GR144577-1), or isotype control antibodies conjugated to Dynabeads (Invitrogen, USA) coated with Protein-A.  After incubation, the immune complexes were collected by centrifugation and washed with the following buffers each for 3 min at 4C;low salt (150 mM NaCl, 50 mM Tris pH8.0, 0.1% SDS), high-salt buffer (500 mM NaCl, 50 mM Tris pH8.0, 0.1% SDS), LiCl buffer (250 mM LiCl, 50 mM Tris pH8.0, 0.5% Na deoxycholate, 1% NP40) and TE (10 mM Tris pH8.0, 1 mM EDTA). The protein-DNA complexes were eluted from the beads in 250 µl elution buffer (1% SDS, 100 mM NaHCO3) at 65C overnight followed by the addition of proteinaseK to 500 µg/ml and incubaton at 56C for 1 hour. Genomic DNA was isolated using QIAquick PCR purification column. ChIP-seq: At least 3 ng of ChIP or input DNA was used for library preparation according to the Illumina ChIP-seq protocol. After end-repair and indexed adapter ligation, fragments were size-selected (average 250 bp) using Agencourt AMPure XP beads (Beckman Coulter, Brea, CA, USA) prior to amplification. The size-selected and amplified fragment libraries were verified on a 2100 Bioanalyzer (Agilent) prior to being pooled (7-10 per lane) and sequenced. FAIRE-Seq:  Cells were fixed with 1% formaldehyde in culture medium for 10 min at room temperature followed by quenching with 0.125 M glycine for 5 min.  Crosslinked chromatin was lysed for 10 minutes sequentially in L1 buffer (50mM Hepes KOH pH 7.5, 140mM NaCl, 1mM EDTA pH 8.0, 10% glycerol, 5% NP40 and 0.25% Triton X-100) followed by L2 buffer (200mM NaCl, 1mM EDTA pH 8.0, 0.5mM EGTA pH 8.0, 1M and Tris pH 8.0). The pellets were resuspended in 300ul of buffer L3 (1mM EDTA pH 8.0, 0.5mM EGTA pH 8.0, 1M Tris pH 8.0, 100mM NaCl, 0.1% Na-deoxycholate and 5mg/ml N-lauroyl sarcosine) and subjected to sonication using a Bioruptor to an average size of 500bp. The soluble DNA fraction was isolated by performing two consecutive phenol:chloroform:isoamylalcohol (25:24:1, pH 8) extractions followed by a chloroform-isoamyl alcohol (24:1) extraction. Precipitated DNA was resuspended in 10mM Tris-HCl (pH 7.4) and incubated with 10 g of RNase A for 1hr. Genomic DNA was isolated using QIAquick PCR purification column. FAIRE-seq: The same protocol was used with the exception that size selected fragments averaged 90 bp.