E. coli CRP Footprint-seq
E. coli CRP Footprint-seq
reaction conditions: 50ng DNA, 1X binding buffer (160mM HEPES (pH 8.0, 20 mM MgCl2, 400 mM potassium glutamate), 0.2 mM cAMP, and varying concentrations of CRP protein in CRP stock buffer(10mM sodium phosphate (pH 6.8), 0.1 mM EDTA, 0.2M NaCl, 50% (w/v) glycerol). Incubate 37C 10 minutes. Then 5ul TDE1 Nextera transposase is added and the reaction is incubated at 37C for 30 more minutes. The DNA from the reaction is purified with a Qiagen MinELute kit. DNA is eluted in 10ul elution buffer. PCR amplify DNA in a 50ul reaction using the Nextera® DNA Library Prep Kit (Illumina, San Diego, CA), following the manufacturer's instructions (2016 version). The kit included custom Nextera PCR primers. Cycling parameters are as follows:  1X 72C for 5 minutes, 98C for 30 seconds 6X 98C for 10 seconds, 63C for 30 seconds, 72C for 1 minute.