GSM2609106: input for "SON TSA-Seq (Ab2) Condition 2 (TSA+Sucrose)"; Homo sapiens; OTHER
Cell lysis and protease digestion were done at 55°C with constant mixing in 10mM Tris, 10mM EDTA, 0.5% SDS and 500µg/ml proteinase K (NEB) for 8-12 hrs (10^7 cells/ml).  NaCl was added for a final 0.2 M concentration and the lysate incubated 20-24 hrs at 65°C with constant mixing to reverse formaldehyde crosslinking.  DNA was extracted 2x using an equal volume of phenol (Fisher)/chloroform/isoamyl alcohol (25:24:1) and 1x with an equal volume of chloroform/isoamyl alcohol (24:1).  Tubes were kept at 55°C for 1h with the cap off to evaporate phenol residues, and then treated with 25 µg/ml RNaseA (Qiagen) at 37o C for 1hr and extracted again.  DNA was then precipitated with 1/10 volume of 3M Na Acetate, pH 5.2, and 2 volumes of 100% EtOH at -20 oC for 20 min – 2 hrs. For each of the TSA-Seq experiment staining conditions, input and Streptavidin pull-down DNA were used to make sequencing libraries, using the Kapa Hyper Prep Kit (Kapa Biosystems).  DNA was blunt-ended, 3’-end A-tailed, and ligated to indexed adaptors with 6nt barcodes. PCR amplification selectively enriched for those fragments with adapters on both ends. Amplification was carried out for 8-15 cycles with the Kapa HiFi polymerase (Kapa Biosystems, Woburn, MA).  An Agilent bioanalyzer DNA high-sensitivity chip (Agilent, Santa Clara, CA) was used to determine library fragment sizes.  Library fragment concentrations were measured by qPCR on a BioRad CFX Connect Real-Time System (Bio-Rad Laboratories, Inc. CA) prior to pooling and sequencing.