biostudies-arrayexpressJames HaycocksEscherichia colihttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-14748ChIP-seq experiments were carried out in Enteroaggregative Escherichia coli 042. Wildtype 042 cells were grown to mid-log phase in LB medium, before being crosslinked with 1% formaldehyde and sonicated to lyse cells and shear DNA. Immunocomplexes were established using anti-FLAG antibodies. The immunocomplexes were then washed before DNA was eluted and libraries prepared for sequencing.biostudies-arrayexpressSequencing - Libraries were sequenced by Azenta Life Sciences, using the sequencing only service. Libraries were sequenced on an Illumina Miseq instrument, in a 2 x 150 bp paired end format.Nucleic Acid Extraction - 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.Library Construction - 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.Sample Collection - 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.Growth Protocol - Escherichia coli 042 cells were grown aerobically to mid-log phase in LB media at 37 degrees C with shaking.OrganizationMINSEQE ScoreAssays and DataMAGE-TAB FilesMetabolomicsUnknownTranscriptomicsGenomicsProteomicsIllumina MiSeqChIP-seqEscherichia coliGenome-wide binding of the cyclic AMP receptor protein in enteroaggregative Escherichia coli suggests a role in orchestrating virulenceJames HaycocksStephen BusbyMunirah AlhammadiMunirah M.T. Alhammadi, Joanne Hothersall, Georgina S. Lloyd, Sophie Titman, Douglas F. Browning, David C. Grainger, Stephen J.W. Busby and James R.J. HaycockfalseChIP-seq analysis of the cAMP binding protein (CRP) in Enteroaggregative Escherichia coli 042ChIP-seq experiments were carried out in Enteroaggregative Escherichia coli 042. Wildtype 042 cells were grown to mid-log phase in LB medium, before being crosslinked with 1% formaldehyde and sonicated to lyse cells and shear DNA. Immunocomplexes were established using anti-FLAG antibodies. The immunocomplexes were then washed before DNA was eluted and libraries prepared for sequencing.2025-07-09T00:00:00Z2025-07-09T14:00:45.114Z2025-01-14T12:56:25.443ZE-MTAB-14748ERP167854EFO_0002944EFO_0004170EFO_0003789EFO_0002692EFO_0005518EFO_0004184