anti-TBP rabbit polyclonal, Santa Cruz Technology, catalog # sc-204X (ChIP-grade), lot # C-0513
Sequenced DNA Library
cells were cross-linked in 1.0% formaldehyde. Cells were then dounced to bread down cells and extract nuclei. Chromatin was sonicated by bioruptor to an average size of 300~500 bp. 300 ul soluble chromatin from ~ 10 million cells was used DNA-protein-complexes were isolated with specific antibodies. Sequencing libraries were prepared following Illumina's instructions: DNA was end repaired using T4 DNA polymerase, DNA polymerase I large fragment (Klenow polymerase) and T4 polynucleotide kinase. A single 3'-A overhang was added to the blunted ends using Klenow 3'-5' exo- polymerase. Adapters with single 3'-T overhangs were ligated to the adenylated fragments, and after a size-selection step using AmPure beads (200-250 bp) the adapter modified DNA was PCR-amplified. Following cluster generation on the flowcell surface, the sequencing libraries were sequenced following Illumina's protocol.