GSM1523615: PBBA2_FAIRE; Homo sapiens; FAIRE-seq
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. 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.