Over-expression of c-Myc-tagged FFRPs allowed enrichment of genomic regions bound by each FFRP in either early or late growth in CDM.
Growth protocol
Halobacterium salinarum NRC-1 strains were batch cultured in complete defined media (CDM) with 19 amino acids in flasks at 37°C with constant shaking (125 rpm).
Extracted molecule
genomic DNA
Extraction protocol
Chromatin immunoprecipitation (IP) was conducted as described in Facciotti, MT, Reiss, DJ, Pan, M, Kaur, A, Vuthoori, M, Bonneau, R, Shannon, P, Srivastava, A, Donohoe, SM, Hood, LE and Baliga, NS. General transcription factor specified global gene regulation in archaea. Proc Natl Acad Sci U S A. 2007;104: 4630-4635. Optimizations of the ChIP protocol are described in Schmid AK, Pan M, Sharma K, Baliga NS.2011. Two transcription factors are necessary for iron homeostasis in a salt-dwelling archaeon. Nucleic Acids Res.39(7):2519-33. Primary antibody is the Covance 9E10 monoclonal antibody (anti-c-myc) from mouse, cat #MMS-150P, and secondary antibody is ECL mouse IgG HRP-linked whole antibody from sheep, Amersham cat #NA931.
Label
Cy3, Cy5
Label protocol
See Baliga NS, Bjork SJ, Bonneau R, Pan M, Iloanusi C, Kottemann MC, Hood L, DiRuggiero J. Systems level insights into the stress response to UV radiation in the halophilic archaeon Halobacterium NRC-1.Genome Res. 2004 Jun;14(6):1025-35.
Over-expression of c-Myc-tagged FFRPs allowed enrichment of genomic regions bound by each FFRP in either early or late growth in CDM.
Growth protocol
Halobacterium salinarum NRC-1 strains were batch cultured in complete defined media (CDM) with 19 amino acids in flasks at 37°C with constant shaking (125 rpm).
Extracted molecule
genomic DNA
Extraction protocol
Chromatin immunoprecipitation (IP) was conducted as described in Facciotti, MT, Reiss, DJ, Pan, M, Kaur, A, Vuthoori, M, Bonneau, R, Shannon, P, Srivastava, A, Donohoe, SM, Hood, LE and Baliga, NS. General transcription factor specified global gene regulation in archaea. Proc Natl Acad Sci U S A. 2007;104: 4630-4635. Optimizations of the ChIP protocol are described in Schmid AK, Pan M, Sharma K, Baliga NS.2011. Two transcription factors are necessary for iron homeostasis in a salt-dwelling archaeon. Nucleic Acids Res.39(7):2519-33. Primary antibody is the Covance 9E10 monoclonal antibody (anti-c-myc) from mouse, cat #MMS-150P, and secondary antibody is ECL mouse IgG HRP-linked whole antibody from sheep, Amersham cat #NA931.
Label
Cy5, Cy3
Label protocol
See Baliga NS, Bjork SJ, Bonneau R, Pan M, Iloanusi C, Kottemann MC, Hood L, DiRuggiero J. Systems level insights into the stress response to UV radiation in the halophilic archaeon Halobacterium NRC-1.Genome Res. 2004 Jun;14(6):1025-35.
Hybridization protocol
According to Agilent protocol.
Scan protocol
ScanArray 5000, using Packard BioChip ScanArray software.
Description
*Sep09_2*.txt Agilent FES file: Cy3=IP, Cy5=WCE. *Sep09_1*.txt Agilent FES file: Cy3=WCE, Cy5=IP (dye flip). Trh7 cMyc-tagged and sampled at late growth, biological replicate 2.
Data processing
Agilent Feature Extraction for spotfinding/quantitation. Normalized log10 intensity of X_INT/Y_INT (IP/WCE) averaged over dye flip between IP and WCE DNA. Second raw data file contains Agilent Feature Extraction file for the dye flip experiment.
