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Sample GSM6427978

Query DataSets for GSM6427978

Status

Public on Mar 18, 2024

Title

Control_2

Sample type

SRA

Source name

Bacterial cells

Organism

Pseudomonas aeruginosa

Characteristics

cell type: Bacterial cells
strain: PAO1
treatment: Control (Without treatment)

Growth protocol

The overnight culture of P. aeruginosa PAO1 propagated in LB was diluted 1:1000 in fresh media and incubated in the presence of Ar5 (16 µg/mL) and PAβN (16 µg/mL) for 17 h at 37 ºC.

Extracted molecule

total RNA

Extraction protocol

After incubation, the 5 mL culture was centrifuged, and the pellet was stored overnight at -80 ºC. The pellet was removed from the freezer, mixed with 1 mL TRIzolTM reagent (Invitrogen), and immediately placed on ice. The solution was subject to bead beating for 45 sec, immediately placed on ice, and centrifuged at 13,000 ×g for 10 min at 4 ºC. The supernatant was transferred to another tube and gently mixed with an equal volume of absolute ethanol (molecular biology grade). Further, the total RNA was extracted using the RNeasy Mini Kit (QIAGEN).
NEB Ultra II directional RNA-Seq Library Prep kit (NEB, Cat# E7760L) protocol was used to prepare libraries for total RNA sequencing. An initial Concentration of 100 ng of total RNA was taken for the assay. First, the total RNA was hybridized with QIAseq® FastSelect™ –5S/16S/23S reagent mixture (Qiagen, Cat# 335927) by heat-fragmenting and cooling the reaction from 75°C to 25°C, followed by Bead clean-up. The cleaned-up ribo-depleted RNA fragments were copied into first-strand cDNA using reverse transcriptase. Second strand cDNA synthesis was performed using DNA Polymerase I and RNase H enzyme. The cDNA fragments were then subjected to a series of enzymatic steps that repair the ends, tails the 3' end with a single 'A' base, followed by ligation of the adapters. The adapter-ligated products were then purified and enriched using the following thermal conditions: initial denaturation of 98°C for 30sec; 13 cycles of - 98°C for 10sec, 65°C for 75sec; final extension of 65°C for 5 mins. The amplified libraries were purified and checked for fragment size distribution on Fragment Analyzer using HS NGS Fragment Kit (1-6000bp) (Agilent, Cat# DNF-474-1000).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Data processing

Read alignment: The paired-end reads are aligned to the reference Pseudomonas aeruginosa genome (https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/006/765/GCF_000006765.1_A M676v1/). Alignment was performed using HISAT2 (2.1.0).
Expression estimation: The aligned reads are used to estimate the gene expression. The raw read counts were estimated using FeatureCount (1.5.2). Read count data were normalized using DESeq2.
Differential expression analysis: The raw read counts were normalized using DESeq2.The ratio of normalized read counts for treated over control was taken as the fold change. Genes were first filtered based on the p-value (<= 0.05). The distribution of these log2 (foldchange) values was found to be normally distributed. Those genes which were found to have -1 <= log2(foldchange) >= 1 were considered as statistically significant.
Assembly: https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/006/765/GCF_000006765.1_A M676v1/
Supplementary files format and content: Microsoft excel files containing normalised read counts, Log2 fold change, Log2 scale values

Submission date

Aug 02, 2022

Last update date

Mar 18, 2024

Contact name

Nisha Mahey

E-mail(s)

nishamahey95@gmail.com

Phone

08427496212

Organization name

CSIR-Institute of Microbial Technology