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Sample GSM5514340

Query DataSets for GSM5514340

Status

Public on Jan 19, 2022

Title

S. maltophilia D457 bacterial culture collected at 0.6 OD600nm - Rep 2

Sample type

SRA

Source name

Bacterial culture

Organism

Stenotrophomonas maltophilia

Characteristics

treatment: untreated

Growth protocol

S. maltophilia were grown overnight in LB broth at 37°C and 250 rpm. This culture was used to inoculate new flasks to reach an optical density at 600 nm (OD600) of 0.01, and the cultures were grown at 37°C until an OD600 of 0.6 was reached. When bacteria grew in the exponential phase (OD600 ≈ 0.6) the transcriptional response of S. maltophilia to a treatment with 16 μg / ml fosfomycin, 2.5 mM PEP or 0.5 GA-3P was evaluated by adding the compounds to the bacterial cultures for 1 hour at 37°C. A culture without treatment was used as the expression control.

Extracted molecule

total RNA

Extraction protocol

Twenty milliliters of each bacterial culture was spun down at 6,000 × g for 3 min at 4°C and immediately frozen on dry ice and stored at −80°C. RNA was isolated from cell pellets using the RNeasy minikit (Qiagen) following the manufacturer’s instructions. To remove the remaining genomic DNA, total RNA samples were treated with DNase I (RNase-Free DNase Set [Qiagen]), and a second digestion was then performed following the Turbo DNA-free (Ambion) protocol. The RNA was purified and concentrated using RNeasy minikit columns. Finally, DNA contamination was checked by PCR with primers 27 and 48. Only RNAs containing no DNA contamination were used for further studies.
Libraries were constructed with Truseq stranded total RNA Library and rRNA depleted with NebNext rRNA depletion kit. Sequencing was conducted by Illumina NovaSeq 6000 sequencing technology using a 150-bp paired-end format and 20 million total reads/sample, using three independent biological replicates of each sample

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

S. maltophilia D457 bacterial culture collected at OD600nm = 0.6

Data processing

Quality check of paired-end short reads (in FASTQ format) was evaluated with FASTQC. Traces of illumina adapters were detected at 3' end so reads were 50 nt cropped in the alignment process
Reads were aligned against S. maltophilia genome (NC_017671) using RNA-STAR (--alignIntronMax 1 --clip3pNbases 50)
Optical and PCR duplicates were identified with 'markduplicates' function of GATK suite
S. maltophilia genes were quantified using featureCounts function from Bioconductor's package Rsubread (strandSpecific=2,isPairedEnd=TRUE,requireBothEndsMapped=TRUE,primaryOnly=TRUE, ignoreDup=TRUE)
Genome_build: NC_017671.1
Supplementary_files_format_and_content: Gene IDs, genome coordinates and raw counts in text-tabulated format

Submission date

Aug 11, 2021

Last update date

Jan 19, 2022

Contact name

Juan Carlos Oliveros

Organization name

CNB, CSIC

Street address

Darwin 3

City

Cantoblanco