# RT-qPCR dataset for "An Hfq-dependent post-transcriptional mechanism fine tunes RecB expression in Escherichia coli" 

doi: https://doi.org/10.7554/eLife.94918.2

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## FOLDER CONTENTS 

This folder contains RT-qPCR data presented in the aforementioned study.  
The data files are in .CSV format and shown according to the order that they appear in the publication figures (the file titles correspond to the figure titles in the publication). 

	Fig 4(S2c): expression of ptrA, recB, recC, and recD mRNAs in delta_hfq cells normalized to the corresponding expression levels in wild-type cells.
	Fig 4(S2d): recB mRNA lifetime in delta_hfq cells normalized to the recB mRNA lifetime in wild-type cells.
	Fig 5(S1b): expression of hfq mRNAs in delta_hfq cells carrying pQE-Hfq plasmid (delta_hfq-pQE-Hfq) normalized to the hfq mRNA levels in wild-type cells. 
	Fig 5(S1c): expression of ptrA, recB, recC, and recD mRNAs in delta_hfq cells carrying pQE80L plasmid (delta_hfq_pQE80L) or pQE-Hfq plasmid (delta_hfq_pQE-Hfq) normalized to the corresponding expression levels in delta_hfq and delta_hfq_pQE80L, respectively.
	Fig 5(S2b): expression of chiX and cyaR RNAs in wild type cells carrying pZA21-ChiX or pZA21-CyaR over-expression plasmid normalized to the corresponding levels in wild-type cells carrying the backbone plasmid pZA21.
	Fig 5(S2c): expression of ptrA, recB, recC, and recD mRNAs in wild-type cells carrying pZA21, pZA21-ChiX or pZA21-CyaR normalized to the corresponding levels in wild-type cells.
	Fig 5(S3b): expression of ptrA, recB, recC, and recD mRNAs in delta_chiX cells normalized to the corresponding levels in wild-type cells.
	
Cq values for each biological replicate for all samples and genes analysed can be found in the "raw_data" files, whilst the data for the fold change analysis using the 2−∆∆Ct method can be found in the "analysed" files.  
Note: some raw data for different figures are combined in the same file as they were obtained in the same experiment. 
The "wild-type" samples were obtained from cells of the MEK65 (MG1655 recB165::halotag) strain. 


## METHODOLOGY 

The qPCR reactions were carried out on a LightCycler 96 (Roche) with a 40 cycles of two-step amplification (95°C, 10s, 4.4°C/s; 60°C, 30s,2.2°C/s) followed by a melting curve (65°C, 60s,2.2°C/s; 97°C, continuous acquisition).  
Initial steps of the data analysis were performed in the LightCycler 96 application software (v1.1.0.1320,Roche) with the built-in protocols:(i) Absolute Quantification (Fit Point) for determination of threshold cycles (Ct) and (ii) High Resolution Melting for evaluation of primer specificity.  
The rrfD gene (5S rRNA) was used as a reference gene to compute a fold-change relative to a control sample with the 2−∆∆Ct method. Outlier samples with the standard deviation std(Ct)>0.3 across technical replicates were removed from the analysis. 
No template controls and no reverse transcriptase controls were performed in each experiment and were deemed as negatives. 
The recB mRNA lifetime measurements were performed in the wild-type and hfq mutant in a time-course experiment where transcription initiation was inhibited with rifampicin. mRNA abundance was quantified with RT-qPCR at t = 0, 1, 2 and 4 min after rifampicin treatment. 
recB mRNA lifetime was calculated from an exponential fit and normalized to the lifetime in the wild-type.