GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM4870590

Query DataSets for GSM4870590

Status

Public on Oct 29, 2020

Title

MIC2_2

Sample type

SRA

Source name

E. coli ATCC8739

Organism

Escherichia coli

Characteristics

source: human feces
strain: ATCC8739
agent: treated with 1/4MIC camphor essential oil

Growth protocol

E. coli ATCC8739, provided by the Strain Preservation Center, was cultured in Luria-Bertani (1% sodium chloride, 1% peptone, and 0.5% yeast extract).

Extracted molecule

total RNA

Extraction protocol

After treating E. coli with lysozyme, total RNA was extracted from E. coli using TRIzol reagent (Invitrogen, USA)
A total amount of 1 μg RNA per sample was used as input material for the RNA sample preparations. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in First Strand Synthesis Reaction Buffer(5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase(RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3’ ends of DNA fragments, Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 370~420 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

MIC2_2

Data processing

Raw data (raw reads) of fastq format were firstly processed through in-house perl scripts. In 1 this step, clean data (clean reads) were obtained by removing reads containing adapter, reads containing ploy-N and low quality reads from raw data. At the same time, Q20, Q30 and GC content the clean data were calculated. All the downstream analyses were based on the clean data with high quality.
Index of the reference genome was built using Hisat2 v2.0.5 and paired-end clean reads were aligned to the reference genome using Hisat2 v2.0.5. We selected Hisat2 as the mapping tool.
FPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene. FPKM, expected number of Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced, considers the effect of sequencing depth and gene length for the reads count at the same time.
Genome_build: Escherichia coli NC_010468.1
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each Sample

Submission date

Oct 28, 2020

Last update date

Oct 29, 2020

Contact name

tian Yu

E-mail(s)

[email protected]

Phone

18379993723

Organization name

Human Aging Research Institute and School of Life Science, Nanchang University, and Jiangxi Key Laboratory of Human Aging