expresses BirA ligase (BirA cells are a negative control- they are equivalent to an IgG only control)
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Briefly, cells were crosslinked for 5 min at room temperature with 1% fresh formaldehyde. Reaction was quenched with glycine (125 mM final concentration) for 5 min at room temperature. Cells were lysed in 0.1% SDS lysis buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH8.0,150 mM NaCl). Chromatin was sonicated to a mean size of 300-500bp. Lysed cells were cleared by centrifugation and added to to 50 μL of Streptavidin Dynal Beads (Life Techniologies, Cat #65601). ψBeads were isolated with a magnet and then washed with SDS Buffer ( 2% SDS) x2, High Salt ( 0.1% Deoxycholate, 1% Triton X-100, 1mM EDTA, 50 mM Hepes, 7.5, NaCl 500 mM), LiCl wash (250 mM LiCl, 0.5% NP-40, 0.5% Deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.0), and TE x2. DNA was eluted and decrosslinked overnight at 65 oC in SDS Elution Buffer (1% SDS, 10mM EDTA, 50 mM Tris-HCl pH 8.0). The next day RNase A and proteinase K were added, and the DNA was precipitated in the presence of glycogen. DNA for downstream applications were quantitated by flourometry (Qubit, Invitrogen), and equal DNA was used for each ChIP-qPCR and normalized to sheared, non-IP’d genomic DNA (Input). The BioScientific NEXTFlex ChIp-seq kit was used.