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Sample GSM7093801

Query DataSets for GSM7093801

Status

Public on Mar 20, 2023

Title

Control strain in the absence of ATc at 2 minutes after rifampicin treatment replicate 1

Sample type

SRA

Source name

Mycolicibacterium smegmatis mc2155

Organism

Mycolicibacterium smegmatis

Characteristics

cell line: Mycolicibacterium smegmatis mc2155
genotype: A hygromycin resistance gene was inserted upstream of, and divergent from, the rne promoter.
treatment: Rifampicin
time: 2 minutes

Treatment protocol

For hypoxia, cultures were diluted to OD 0.01, sealed in bottles with a 1:1 airspace:culture ratio, and incubated with shaking for 18 hours. Rifampicin was then injected by a syringe to a final concentration of 150 µg/mL and cultures were harvested by freezing in liquid nitrogen at the indicated timepoints.
For non-hypoxic samples, strains were grown to OD 0.8-0.9, with or without addition of ATc 8 hrs prior as indicated. Rifampicin was then added to a final concentration of 150 µg/mL and cultures were harvested by freezing in liquid nitrogen at the indicated timepoints.

Growth protocol

Strains were growth in 7H9 supplemented with ADC, glycerol, and Tween-80.

Extracted molecule

total RNA

Extraction protocol

Pellets were resuspended in Trizol and lysed by bead-beating. RNA was extracted with a Zymo Direct-zol kit, treated with DNase, and purified with a Zymo Clean and Concentrate kit.
Libraries were constructed by the Broad Institute Microbial 'Omics Core using the protocol described in https://doi.org/10.1038/nmeth.3313
Ligation of 3' and 5' adapters to sheared RNA to construct paired-end Illumina RNAseq libraries with TruSeq-style adapters

Library strategy

OTHER

Library source

transcriptomic

Library selection

other

Instrument model

Illumina NextSeq 500

Data processing

For mRNA degradation profiling, reads were aligned to the reference genome using BWA-MEM v0.7.17 with default parameters.
For mRNA degradation profiling, the alignments were processed for each strand using SAMtools v1.10.
For mRNA degradation profiling, raw coverage of each coordinate was calculated using BEDTools v2.29.1.
For mRNA degradation profiling, the coverage for each gene was calculated as the summation of the coverage of its coordinates using custom Python script.
For mRNA degradation profiling, the raw coverage were nomalized by the library size, relative qPCR expression levels and gene length using custom Python script.
For RNaseE cleavage site analysis, reads were filtered using Trimmomatic v0.39 with the parameters "SLIDINGWINDOW:4:20 MINLEN:25".
For RNaseE cleavage site analysis, the filtered reads aligned to tRNA and rRNA were removed using Bowtie2 v2.4.5 with the parameter "--very-sensitive".
For RNaseE cleavage site analysis, the remaining reads were aligned to genome using Bowtie2 v2.4.5 with the parameter "--very-sensitive".
For RNaseE cleavage site analysis, the alignments were filtered using SAMtools v1.16.1 with the parameter "-q 10 -F 260".
For RNaseE cleavage site analysis, raw strand specific coverage of each coordinate was calculated using SAMtools v1.16.1 and BEDTools v2.30.0.
For RNaseE cleavage site analysis, the single-nucleotide coverage for each gene was normalized by the summed coverage of that gene.
Assembly: NC_008596.1
Supplementary files format and content: mRNA_halfLife_normalized_log2_coverage.txt is a text file including log2 normalized gene coverage for all replicates of all time points and conditions.
Supplementary files format and content: RNaseE_cleavageSite_normalized_coverage.txt is a text file including normalized single-nucleotide coverage and log2 coverage ratio between rne knockdown and control for qualified nucleotide coordinates, along with the annotations for each coordinate.

Submission date

Mar 13, 2023

Last update date

Mar 20, 2023

Contact name

Scarlet Shell

E-mail(s)

sshell@wpi.edu

Organization name

Worcester Polytechnic Institute

Department

Biology & Biotechnology