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Sample GSM4214743

Query DataSets for GSM4214743

Status

Public on Jul 10, 2020

Title

RNA-seq from dmazEF strain

Sample type

SRA

Source name

bacterial cell

Organism

Staphylococcus aureus

Characteristics

strain: dmazEF

Growth protocol

S. aureus HG003 and S. aureus HG003 ΔmazEF were electroporated with anhydrotetracycline (ATc) inducible pRAB11 and pRAB11-mazF constructed vector. Strains carrying the pRAB11 vector were always grown in presence of chloramphenicol (15 µg/mL). Overnight cultures were diluted 1/1000 in 10 mL of Mueller-Hinton broth (cation adjusted, Difco) and incubated at 37ºC with shaking. At OD600 0.3, ATc was added to a final concentration of 0.2 µM. At each time point (0, 15, 30, 60 minutes postinduction) the samples were subject to 10-fold serial dilutions in 96-well plates using saline solution (0.9% NaCl), 10 µL aliquots were spotted in Mueller-Hinton (Difco) agar plates and incubated at 37ºC overnight. Colonies were then counted.

Extracted molecule

total RNA

Extraction protocol

Overnight cultures were diluted 1/50 in 20 mL of Mueller-Hinton broth and incubated at 37ºC without shaking until OD600 0.3. ATc was added to a final concentration of 0.2 µM and incubated at 37ºC during 10 min. 8 mL of bacterial culture were immediately transferred to 40 mL of ice-cold ethanol:acetone (1:1), and centrifugated at 5000 rpm and 4ºC during 10 min. The supernatant was discarded, and pellet was washed with 1X TE. The pelleted cells were treated with lysostaphin (200 µg/mL) and RNasin Plus (Promega) in a final volume of 100 µL then placed in a heat block at 37ºC during 10 min. RNA extractions were done on the same day using the ReliaPrep (Promega) following the manufacturer’s instructions. Total RNA was frozen and kept at -80ºC.
TruSeq mRNA stranded kit + bacterial Ribo-Zero RNA removal kit

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Data processing

nEMOTE reads were processed with the R package EMOTE v0.2 (https://github.com/pradosj/EMOTE). The package check the reads and parse them to extract the mapping sequence as well as the UMI sequence; it then aligns the reads on the genome (with Rbowtie package) and quantify the number of read with unique UMI starting at each genomic position.
The genomic positions with less than 4 UMI in ΔmazEF pRAB11-mazF-1_+PNK or ΔmazEF pRAB11-mazF-2_+PNK were not further considered
A beta-binomial model is used to estimate the probability that +PNK condition is significantly enriched compared to the corresponding -PNK condition. The p-values obtained for the 2 replicates are combined with Fisher's method, and the FDR computed to correct for multiple testing.
To determine confident 5’-OH ends generated by MazF, each genomic position had to match the following criteria: an FDR < 0.1 for ΔmazEF pRAB11-mazF condition, and an FDR > 0.1 in ΔmazEF condition
Genomic position falling into a rRNA feature are processed separately
Remaining mazF cleavages are further annotated with genomic informations (DNA sequence arround the position, nearest gene)
RNA-seq reads are aligned on the reference genome with "bwa mem" command and "samtools" to generate files in BAM format. The BAM files are further processed to quantify the number of read in genes with the method "summarizeOverlaps(mode='IntersectionStrict',inter.feature=FALSE)" from R package GenomicAlignments. Quantification arround the cleavage sites are obtained with the method "coverage()" of the R package "GenomicAlignments"
Genome_build: NC_007795.1

Submission date

Dec 11, 2019

Last update date

Jul 10, 2020

Contact name

Julien Prados

E-mail(s)

[email protected]

Phone

+41 22 37 95 396

Organization name

University of Geneva