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Sample GSM4214741

Query DataSets for GSM4214741

Status

Public on Jul 10, 2020

Title

Second sample prepared using nEMOTE protocol from dmazEF+pF strain, without PNK

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.
nEMOTE as in Kirkpatrick, 2016. Briefly, mono-phosphorylated RNA was digested from 8 µg of high-quality total RNA with XRN-1. XRN-1 was removed with a phenol - chloroform - isoamyl alcohol 25:24:1 (Sigma) extraction, and the aqueous phase was recovered using MaXtract High Density (Qiagen) tubes. The recovered RNA was split into two pools, one pool was treated with PNK (+PNK) and the other was subject to the same treatment without PNK (-PNK). Rp6 RNA oligonucleotide was ligated to the 5’-phosphorylated RNA followed by an ethanol precipitation. Reverse transcription was carried out using semi-random primer DROAA and the cDNA was purified using Wizard SV Gel and PCR clean-up system (Promega). Second-strand synthesis and barcode incorporation were performed using Q5 Hot Start High Fidelity DNA polymerase (New England Biolabs). The PCR products were purified, mixed and size-selected between 300 – 1000 bp. The sample was then quantified and sequenced in an Illumina HiSeq 2500 sequencer using 50 bp single-read cycle.

Library strategy

OTHER

Library source

transcriptomic

Library selection

other

Instrument model

Illumina HiSeq 2500

Data processing

Library strategy: nEMOTE-seq
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