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Sample GSM3314428

Query DataSets for GSM3314428

Status

Public on Sep 27, 2018

Title

Bl-04_LOG_8_2

Sample type

SRA

Source name

Bacterial culture

Organism

Bifidobacterium animalis subsp. lactis

Characteristics

strain: Bl-04
tetracycline conc.: 8 ug/mL
growth condition: LOG (ISO method undiluted)

Treatment protocol

Tetracycline exposure

Growth protocol

Method described in ISO (2010). "ISO 10932:2010 (IDF 223:2010). Milk and milk products -- Determination of the minimal inhibitory concentration (MIC) of antibiotics applicable to bifidobacteria and non-enterococcal lactic acid bacteria (LAB)." Int. Organ. Stand. (ICS: 67.100.01.): 1–31.

Extracted molecule

total RNA

Extraction protocol

After reaching early log phase, cells were centrifuged for 10 min. at 4,063 x g. The supernatant was removed and 1 mL of TRIzol (p/n: 15596026, Thermo) was added. The pellet was re-suspended by vortexing and the tubes were immediately frozen at -80°C. The cell pellets were later thawed, transferred to a Lysing Matrix B 2mL tube (p/n: 116911050, MPBio, Santa Ana, CA), and disrupted using a Mini-Beadbeater. The lysate was subjected to a chloroform organic extraction and followed by purification using RNeasy Mini Kit (p/n: 74104, Qiagen, Hilden, Germany).
Quality checks and quantifications of the isolated RNA were carried out on the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Ribosomal RNA was removed prior to library construction using Ribo-Zero rRNA Removal Kit Gram-Positive Bacteria (p/n: MRZGP126, Illumina). Stranded cDNA libraries were prepared using TruSeq Stranded mRNA Kit (p/n: 20020594, Illumina), quantitated by Agilent TapeStation, pooled quimolarly, and sequenced on one flowcell lane for 75 cycles using paired-end 75 basepair sequencing on Illumina 2500 HiSeq Rapid Cluster Kit v. 2 (p/n: PE-402-4002, Illumina) and HiSeq Rapid SBS Kit v. 2 (p/n: FC-402-4021, Illumina).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

Data processing

Paired-end reads were imported and mapped to the B. lactis Bl-04 genome (NC_012814.1) using the “Geneious for RNA” Mapper with default settings in Geneious v. 11.0.4
Transcript levels in the resulting assemblies were calculated using the “Calculate Expression Levels” function, which enabled the comparison of replicates and experiments using the “Compare Expression Levels” with the DESeq2 method and parametric fit type.
The assemblies were exported as BAM files and imported into ArrayStar v. 1.2 (DNAStar, Madison, WI), processed using QSeq, and normalized by reads per kilobase of transcript per million mapped reads (RPKM).
Regression analyses of the RNA data were made in ArrayStar software using the Student’s t-test with FDR correction. Statistical analyses between sets of samples were analyzed using DESeq2 method as above. Differences in expression were considered significant if the Absolute Confidence (-Log10 adjusted p-value) was +1.00, and the Log2 ratio was at + 1.00, representing p < 0.05 and a > 2x fold change, respectively, after normalization.
Genome_build: CP001515
.bam sequence alignment using Geneious v. 11.0.4
Supplementary_files_format_and_content: raw counts

Submission date

Jul 30, 2018

Last update date

Sep 27, 2018

Contact name

Wesley w. Morovic

E-mail(s)

wesley.morovic@dupont.com

Phone

608-395-2819

Organization name

DuPont Nutrition & Health