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Sample GSM8077108

Query DataSets for GSM8077108

Status

Public on Jul 16, 2024

Title

Strain CF13, rep 2

Sample type

SRA

Source name

Enterobacteria

Organism

Citrobacter freundii

Characteristics

strain: CF13
cell type: Enterobacteria
genotype: WT

Growth protocol

We grew the cells in LB medium with continuous shaking. When cultures reached a turbidity of 0.5 at 600 nm, we collected the cells at 4ºC, pelleted them by centrifugation at 4ºC 12,000 g for 1 min and immediately froze them at -70ºC.

Extracted molecule

total RNA

Extraction protocol

We purified the total RNA from each sample using the NZY Total RNA Isolation kit (NZYTECH). Then, we determined RNAs concentration using the Qubit RNA Broad-Range Assay following the manufacturer's instructions. Additionally, the quality of the RNA was examined using the Tape-Station system (Agilent).
We ribodepleted the RNA and sequenced it at the Wellcome Trust Centre for Human Genetics (WTCHG; Oxford, UK) using the Illumina's NovaSeq6000 platform, resulting in >8 million reads per sample.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

CF13.2
CF13_featureCounts.tsv
CF13_DE_results_raw.tsv

Data processing

We trimmed and adapter-removed the reads with Trim Galore v0.6.4 (-q 20 --length 50 --illumina). We assessed the quality of the reads before and after trimming with FastQC v.0.11.9 and MultiQC v1.11.
We first identified in the genomes all copies of all IS families annotated by PGAP. Then, we masked the sequences of all but one copy of ISs per family (leaving the unmasked single copy preferentially in the chromosome or in the largest plasmid) from the reference genomes, using the bedtools maskfasta tool v2.27.1. This way, reads belonging to an IS family (like IS1) can map with low ambiguity to one region of the genome.
After re-annotating the masked genomes with PGAP v2021-07-01.build5508, we mapped the trimmed reads to their corresponding reference genome with BWA-MEM v0.7.17.
We inspected the mappings to confirm the appropriate presence or absence of pOXA-48 in the replicates. A replicate in K147c1 and in CF13c1 (cured from pOXA-48) actually carried the plasmid, so they were included as an additional replicate of the pOXA-48-carrying strains. The third replicate of C063p had a mutation in pOXA-48 (near the gene repA) that caused an increased plasmid copy number, unlike the other two replicates. Thus, we removed this replicate from the analysis.
We obtained the raw counts of reads mapping to each genomic feature (including CDS, ncRNA, tRNA, tmRNA, antisense RNA, RNase P and SRP RNA) with featureCounts from the Rsubread v2.14.2 package.
We performed the differential expression analysis from raw counts using DESeq2 v1.40.1 by comparing the pOXA-48-carrying strain against the pOXA-48-free strain.
Assembly: SAMN32542096, SAMN32542113, SAMN32542116, SAMN32542119, SAMN32542124, SAMN32542126, SAMN14640123, SAMN14640085, SAMN14640188, SAMN14640201
Supplementary files format and content: tab-delimited text file includes raw counts for each sample (<strain>_featureCounts.tsv)
Supplementary files format and content: tab-delimited text files include log2FoldChanges, padj and other values for each differential expression analysis (<strain>_DE_results_raw.tsv)

Submission date

Feb 13, 2024

Last update date

Jul 16, 2024

Contact name

Alvaro San Millan

E-mail(s)

[email protected]

Organization name

Centro Nacional de Biotecnología-CSIC

Department

Biotecnología Microbiana