ChIP-seq: Cells were cross-linked by adding formaldehyde to the culture medium to a final concentration of 1% (8 min at RT). Cross-linking was stopped by addition of glycine to a final concentration of 125 mM. Cells were washed twice with PBS and lysed in SDS buffer: 100 mM NaCl, 50 mM Tris HCl (pH 8.1), 5 mM EDTA (pH 8), 0.5% SDS, and protease inhibitors. Chromatin lysate was then pelleted and resuspended in IP buffer (100 mM NaCl, 100 mM Tris HCl at pH 8.1, 5 mM EDTA at pH 8, 0.3% SDS, 1.7% Triton X-100), where it was directly sonicated prior to overnight incubation with antibodies . ChIP-seq: Libraries were prepared according to HT-ChIP-Seq Library Preparation Protocol. Briefly, DNA was end-repaired using a combination of T4 DNA polymerase and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (3´→ 5´ exo minus) and dATP to yield a protruding 3- 'A' base for ligation of indexed oligo adaptors which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA was PCR amplified with amplification primers for 14 cycles. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols. BLISS: Cells were fixed for 10 min in 4% formaldehyde at room temperature (RT). After permeabilization, cells were incubated in a blunting reaction mix (NEB, cat. no. E1201L) for 1 h at RT, followed by in situ DSB ligation in a T4 DNA ligase reaction mix (NEB, cat. no. M0202M) for 16–18 h at 16 °C. The next day, genomic DNA (gDNA) was in situ fragmented by incubating the samples with HaeIII (NEB, cat. no. R0108L) for 3 h at 37 °C. Afterwards, the cells were scraped off the coverglass, and gDNA purified using proteinase K (NEB, cat. no. P8107S). Purified gDNA was linearly amplified using the T7 RNA polymerase (Thermo, cat. no. AM1334). BLISS: Libraries were prepared using a modified Illumina TruSeq Small RNA Library Prep Kit (RS-200-0012). A detailed, step-by-step BLISS protocol can be found at https://doi.org/10.1101/091629.
GEO Accession
GSM2442946