ChIP-seq analysis of the cAMP binding protein (CRP) in Enteroaggregative Escherichia coli 042
ChIP-seq analysis of the cAMP binding protein (CRP) in Enteroaggregative Escherichia coli 042
Escherichia coli 042 was isolated from Lima, Peru (Nataro JP, Baldini MM, Kaper JB, Black RE, Bravo N, Levine MM. J Infect Dis. 1985;152(3):560-565. Escherichia coli 042 cells were grown aerobically to mid-log phase in LB media at 37 degrees C with shaking. ChIP-seq was performed using a method based on those used by Middlemiss et al. (Microbiology (Reading). 2023 May;169(5):001330) and Zanin et al. (Commun Biol. 2023 Nov 2;6(1):1112). Escherichia coli 042 cells were sub-cultured from overnight cultures (using a 1/100 dilution) into fresh LB media and were grown with shaking at 37 degrees C to mid-log phase, before being crosslinked by the addition of 1% formaldehyde (final concentration) for 20 minutes. The reaction was quenched with the addition of 0.5 M glycine (final concentration). Cells were washed in 1x TBS, before resuspension in FA-1 buffer (150 mM NaCl) containing 2 mg/ml lysozyme. Cells were incubated at 37 degrees C for 30 mins, before sonication was carried out using a Bioruptor instrument (30 cycles, 30 secs on, 30 secs off, high intensity) to lyse cells and shear DNA. Lysates were clarified by centrifugation before storage at -80 degrees C until required. For anti-CRP experiments, 150 μg of chromatin was diluted to 700 μl with FA-1 and 2 μl 1D8D9 anti- E. coli CRP antibody (BioLegend), these were incubated on a rotating wheel at 4 degrees C overnight. For mock experiments, 2 μl FA-1 was added in lieu of the antibody. The following day, 15 μl each of protein A and protein G Dynabeads (Invitrogen, pre-washed in FA-1 buffer) were added and left to incubate for 1 hour at 4 degrees C. Immunocomplexes were then washed 6 times with FA-1 buffer (150 mM), the subsequently with FA-2 buffer, FA-3 buffer and finally 1x TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). DNA was eluted by the addition of 195 μl elution buffer, and incubation at 65 degrees C for ten minutes. De-crosslink was done through addition of 0.25 mg proteinase K (Sigma) and incubation at 42 degrees C for 10 minutes, then a further incubation at 65 degrees C for 4 hours. DNA was purified using a Qiagen PCR purification kit. Sequencing libraries was made using an NEBnext Ultra II DNA kit, following the manufacturer's instructions. Libraries were checked using a Agilent Tapestation instrument, and were quantified by qPCR using an NEBnext library quantification kit. Libraries were pooled in an equimolar ratio prior to sequencing.