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Sample GSM7106016

Query DataSets for GSM7106016

Status

Public on Aug 07, 2023

Title

wild-type, biological replicate 5, 1 mM K2HPO4 (Pi)

Sample type

SRA

Source name

wild-type, biological replicate 5, 1 mM K2HPO4 (Pi)

Organism

Salmonella enterica

Characteristics

cell line: 14028s
cell type: Planktonic
genotype: wild-type
treatment: 1 mM K2HPO4
time: 35 minutes

Treatment protocol

35 minutes in MOPS medium lacking a phosphorus source.
35 minutes in MOPS medium containing 1 mM K2HPO4.

Growth protocol

Four independent biological replicates of wild-type and phoB strains were grown in MOPS medium supplemented with 1 mM K2HPO4 to an OD600 of 0.4-0.45. Bacterial cultures were subsequently split into two equal fractions of approximately 10 OD600 units, and were labeled either as “-P” or “+Pi”. -P-treatment cells were washed twice (6,000 X g, 5 min) with MOPS medium lacking K2HPO4 (or any other P source). Conversely, +Pi-treatment cells were washed in MOPS medium supplemented with 1 mM K2HPO4. Bacterial cultures were then grown for 35 min to induce the Pi-starvation response. Cultures were then treated with RNAprotect (Qiagen) for 5 min at RT to stabilize mRNA, and then collected by centrifugation (6,000 X g, 5 min).

Extracted molecule

total RNA

Extraction protocol

Cultures were then treated with RNAprotect (Qiagen) for 5 min at RT to stabilize mRNA, and then collected by centrifugation (6,000 X g, 5 min). Total RNA was isolated by hot-phenol extraction, and further purified using the RNeasy Kit (Qiagen) with on-column DNase I treatment. The eluates were treated with Turbo DNase (Invitrogen) for 30 min before a second round of purification using the RNeasy Kit. rRNA was depleted using the NEBNext® rRNA Depletion kit (Bacteria) and purified from the enzymatic reactions using RNAClean XP magnetic beads (Beckman Coulter) following manufacturer’s instructions. RNA quality and ribosomal depletion was assessed using BioAnalyzer (Agilent).
RNA-seq libraries were made and sequenced by the Genomics Core Facility at Penn State University.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

NextSeq 550

Description

total RNA, ribosomal RNA-depleted

Data processing

Untrimmed reads were mapped to the Salmonella enterica serovar Typhimurium 14028s reference genome (plasmid NC_016855.1 and chromosome NC_016856.1) using default settings in the SeqMan NGen (DNASTAR v17.0.2.1) software.
Assembly files were analyzed in Geneious Prime v11 software. RPKM, FPKM, and TPM read counts were calculated using Geneious prior to differential expression analysis using the DeSeq2 Geneious plugin with a false discovery rate of 0.1.
Genes with log 2-fold changes above and adjusted p-values less than 0.01 were considered significantly different.
Assembly: ASM824478v1
Supplementary files format and content: tsv files containing annotated genes, their corresponding locus tags, differential expression values and associated statistic tests and p-values for differentail expressions. Four comparisons were performed: assembled reads from phoB mutant grown in medium lacking phosphorus (P) vs. phoB mutant grown in medium containing phosphate 1 mM K2HPO4 (Pi) for the plasmids and chromosome (phoB_no P vs Pi_NC_016855.tsv and phoB_no P vs Pi_NC_016856.tsv files, respectively), and assembled reads from wild-type grown in medium lacking P vs. wild-type grown in medium containing Pi for the plasmids and chromosome (WT_no P vs Pi_NC_016855.tsv and WT_no P vs Pi_NC_016856.tsv files, respectively).

Submission date

Mar 20, 2023

Last update date

Aug 07, 2023

Contact name

Mauricio Henriques Pontes

E-mail(s)

mpontes@pennstatehealth.psu.edu, mxh1355@psu.edu

Organization name

Penn State University

Department

Pathology