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Sample GSM4551485

Query DataSets for GSM4551485

Status

Public on Aug 04, 2023

Title

LT2_Fur_KO_Fe_2

Sample type

SRA

Source name

bacteria cells

Organism

Salmonella enterica

Characteristics

strain: Typhimurium LT2
medium: glucose M9 minimal
phase: mid-log phase
genotype: Fur deletion

Treatment protocol

For iron-replete condition, M9 minimal media was supplemented with 0.1 mM FeCl2 ; for iron-depleted condition, M9 minimal media was supplemented with 0.2 mM 2,2’-dipyridyl at early-log phase and continue to culture at 37 oC for additional 2h with vigorous agitation; for regulatory condition, M9 minimal media was supplemented with 1 mL trace element solution (100X) containing 1 g EDTA, 29 mg ZnSO4.7H2O, 198 mg MnCl2.4H2O, 254 mg CoCl2.6H2O, 13.4 mg CuCl2, and 147 mg CaCl2.

Growth protocol

glycerol stocks of E. coli and S. Typhimurium LT2 strains were inoculated into M9 minimal media with 0.2% (w/v) glucose. The culture was incubated at 37 oC overnight with agitation, and then was used to inoculate the fresh media (1/200 dilution). The volume of the fresh media was 150 mL for each biological replicate. The fresh culture was incubated at 37 oC with agitation to the mid-log phase (OD600 ≈ 0.5).

Extracted molecule

total RNA

Extraction protocol

Three milliliters of cells from mid-log phase culture were mixed with 6 ml RNAprotect Bacteria Reagent (Qiagen), mixed immediately by vortexing for 5 seconds, incubated for 5 minutes at room temperature, and then centrifuged at 5000g for 10 minutes. The supernatant was decanted and any residual supernatant was removed, then samples were frozen at -80℃. Next, RNA was harvested using RNeasy Plus Mini kit (Qiagen) in accordance with the manufacturer’s instruction. Illumina Ribo-Zero rRNA removal kit (bacteria) (MRZMB126) was used with 1 ug of total RNA for the removal of rRNA procedure. Then KAPA RNA HyperPrep kits(Cat.No.08098107702) was used with rRNA-depleted RNA samples for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using standard Illumina protocols

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Description

LT2

Data processing

The base calling was done using Illumina’s Real-Time Analysis (RTA) v2.7.7; data was converted to fastq and demultiplexed using Illumina’s bcl2fastq2 Conversion Software v2.20;
Sequence reads generated from RNA-seq were mapped onto each reference genome using bowtie with the maximum insert size of 2000 bp, and 2 maximum mismatches after trimming 3 bp at 3’ ends
SAM files generated from bowtie, then, were then used for DESeq(https://bioconductor.org/packages/release/bioc/html/DESeq.html)
Cufflinks was run with default options with the library type of dUTP RNA-seq and the default normalization method (classic-fpkm)
Genome_build: The reference genomes used in this study are shown as below, MG1655 = NC_000913.2 / W3110 = NC_007779.1 / W = NC_017635.1 , NC_017636.1 , NC_017637.1 / KO11FL = NC_017660.1 , NC_017661.1 / 042 = NC_017626.1 , NC_017627.1 / BL21 = NC_012971.2 / Crooks = NC_010468.1 / Sakai = NC_002695.1 , NC_002127.1 , NC_002128.1 / CFT073 = NC_004431.1 / LT2 = NC_003197.2 , NC_003277.2
Supplementary_files_format_and_content: tab-delimited text files containing TPM values for each sample

Submission date

May 13, 2020

Last update date

Aug 04, 2023

Contact name

Ye Gao

E-mail(s)

yeg002@ucsd.edu

Organization name

UCSD

Street address

9500 Gilman Dr.