Comment[ArrayExpressAccession]	E-GEOD-46120
MAGE-TAB Version	1.1									
Public Release Date	2014-05-22
Investigation Title	Small RNAs and RNA chaperone Hfq in enterohemorrhagic E. coli									
Comment[Submitted Name]	Small RNAs and RNA chaperone Hfq in enterohemorrhagic E. coli
Experiment Description	This SuperSeries is composed of the SubSeries listed below. Refer to individual Series									
Term Source Name	ArrayExpress	EFO
Term Source File	http://www.ebi.ac.uk/arrayexpress/	http://www.ebi.ac.uk/efo/efo.owl								
Person Last Name	Tree									
Person First Name	Jai 									
Person Mid Initials	Justin									
Person Email	jtree@staffmail.ed.ac.uk									
Person Affiliation	University of Edinburgh									
Person Phone	+44 0131 6519229									
Person Address	The Roslin Institute, University of Edinburgh, The Roslin Insitute, Edinburgh, Scotland, United Kingdom									
Person Roles	submitter									
Protocol Name	P-GSE46120-1	P-GSE46120-5	P-GSE46120-6	P-GSE46120-2	P-GSE46120-8	P-GSE46120-4	P-GSE46120-10	P-GSE46120-9	P-GSE46120-3	P-GSE46120-7
Protocol Description	LOWESS was used to normalise data generated by GenePix using Genespring GX 7.3.1.  Flags: -50, -75, -100. ID_REF =  VALUE =  Lowess normalized log2 (Alexa 555/Alexa 647) test/reference [average of 3 replicates] PRE_VALUE =  Lowess normalized (Alexa 555/Alexa 647) test/reference [average of 3 replicates]	Total RNA was labelled using the Superscript Plus indirect labelling system as per manufacters instructions	The slides were incubated for 20 min in prewarmed Universal Pre-Soak solution at 42 °C and washed twice in 0.1_SSC, 0.1% SDS for 30 s at room temperature. Slides were immediately transferred into prewarmed prehybridization solution (5_ SSC, 0.1% SDS, and 0.1% bovine serum albumin) and incubated for 2-4 h at 42 °C. The microarray slides were finally washed at room temperature once in 0.1x SSC, 0.1% SDS for 1 min and twice in 0.1x SSC for 30 s, briefly dipped in water and then ethanol, and dried by centrifugation for 5 min at 800g. For each experiment, equal quantities Alexa fluor 555 and 647-labeled cDNA were added to hybridization solution containing 25% formamide, 10 micro grams of bovine serum albumin (fraction V) per ml, 5x SSC (1x SSC is 0.15 M NaCl, 0.015 M sodium citrate), 0.1% SDS, 8 micro grams of poly(A), and 1x Denhardt's solution. The cDNA probes were denatured at 95 °C for 3 min and hybridized for 16 h at 42 °C. After hybridization was complete, the slides were washed in 2_SSC, 0.1% SDS at 42 °C for 2 min in 0.1x SSC, 0.1% SDS for 2 min at room temperature, and finally twice in 0.1x SSC for 2 min at room temperature. The microarray slides were dried by centrifugation for 5 min at 800g.	10ml of cells were incubated in an equal volume of RNAprotect and centrifuged.	Cells were crosslinked with 1800mJ of UV-C and collected by centrifugation. Cell were pelleted, resuspended in PBS, and divided into 1g aliquots that were snap frozen in liquid nitrogen.	Total RNA was extracted using an Qiagen RNeasy mini Kit as per manufacter instructions.	Cells were lyzed by vortexing with zirconia beads in lysis buffer. Hfq.RNA complexes were purified over M2 anti-FLAG resin, trimmed with RNase A/T1 and repurified under denaturing conditions using Ni-NTA resin. RNA.Hfq complexes were seperated by SDS-PAGE, transferred to a nitrocelluose membrane and extracted. Protein was removed by Prrteinase K digestion and RNAs purified by phenol:chloroform extraction. Extracted RNAs were converted to cDNA and PCR amplified. PCR products between ~150 and 250 bp were gel extracted and submitted for Solexa sequencing.	Strains were intially grown in LB for 16hrs and then diluted (1/100) into appropriate media and grown to and OD600 of ~0.8 at 37 degrees C.	Strains were cultured overnight in LB media at 37 degrees and diluted 1/100 into MEM-HEPES media supplemented with 0.1% glucose and 250nM FeCl3. Cell were harvested at OD600 0.8	Slide was scanned at 532 and 630 nm by using a Genepix 4000A scanner (Axon Instruments, Union City, CA).
Protocol Type	normalization data transformation protocol	labelling protocol	hybridization protocol	sample treatment protocol	sample treatment protocol	nucleic acid library construction protocol	nucleic acid library construction protocol	growth protocol	growth protocol	array scanning protocol
Experimental Factor Name	STRAIN	MEDIA	GENOTYPE	ORGANISM	RNA CHAPERONE HFQ					
Experimental Factor Type	strain	media	genotype	organism	rna chaperone hfq					
Comment[SecondaryAccession]	GSE46120									
Comment[GEOReleaseDate]	2014-05-22									
Comment[ArrayExpressSubmissionDate]	2013-04-16									
Comment[GEOLastUpdateDate]	2014-05-23									
Comment[AEExperimentType]	transcription profiling by array									
Comment[AEExperimentType]	RNA-seq of coding RNA									
SDRF File	E-GEOD-46120.hyb.sdrf.txt	E-GEOD-46120.seq.sdrf.txt