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Sample GSM1569064

Query DataSets for GSM1569064

Status

Public on Jul 01, 2015

Title

CSS rep1

Sample type

SRA

Source name

cold smoked slamon

Organism

Listeria monocytogenes

Characteristics

strain: H7858
inoculated on: CSS
growth stage: late log phase

Growth protocol

Defined media cultured L. monocytogenes was inoculated on cold smoked salmone (CSS) and in modified BHI broth (MBHIB) at approximately 1x106 CFU/ml or g and incubated for 7 days to late-log phase at 7oC before RNA extraction. Growth phase was determined by growth parameters generated by Buchannan model and growth curve information obtained from pilot growth experiments. Samples for RNA preparation were collected when the average cell density of collected L. monocytogenes samples were 8.17 ± 0.16 log(CFU/ml) for CSS and 8.28 ± 0.21 log(CFU/g) for MBHIB.

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted by using RNA protect, proteinase K, and lysozyme, followed by Tri reagent with bead-beating. Total RNA was incubated with DNase in the presence of RNasin to remove remaining DNA.
Preparation of cDNA fragment libraries was performed using the ScriptSeqTM Complete Kit (Bacteria)-Low Input (Epicentre, Madison, WI), which is composed of Ribo-ZeroTM rRNA Removal Reagents (Bacteria)-Low Input, Magnetic Core Kit-Low Input, and ScriptSeqTM v2 RNA-Seq Library Preparation Kit.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

Total RNA was treated with Ribo-ZeroTM rRNA Removal Reagents (Bacteria)-Low Input for removal of 16S and 23S ribosomal RNA

Data processing

Raw data was generated by the Illumina pipeline software v1.8
the sequence reads were aligned to a H7858 pseudochromosome out using the Burrows-Wheeler Aligner (BWA), which allowed up to 2 mismatches per read. BWA parameters were set to include only perfect matches and map once reads, i.e., uniquely mapped reads are retained. The output was a SAM files for each sample. Used SAMTOOLS (Li, et al., PMID 1950593) to sort and index the SAM files obtained from BWA and convert them to BAM format. BAM files were converted to strand-specific base count files. Coverage at each base position along the chromosome was calculated by enumerating the number of reads that aligned to a given base for each DNA strand separately.
Differential expression of genes in different strains was statistically assessed using the BaySeq method implemented in the BaySeq 1.16.0 package available from Bioconductor. Genes were considered differentially expressed if they showed a false discovery rate (FDR) < 0.05 and a fold change (FC) ≥ 2.5 (meaning genes were upregulated on CSS) or FC ≤ 0.2 (meaning genes were downregulated on CSS).
Genome_build: H7858 pseudochromosome was constructed based on contigs available at NCBI Genbank under Accession: NZ_AADR00000000.1 / GI: 47094674
Supplementary_files_format_and_content: Excel spreadsheet (.xlsx) file. Raw count data and normalized count data (from BaySeq output) for each coding gene in H7858. Locus names in H7858, F2365, EGD-e and 10403S are also provided as well as the function of the protein encoded. BaySeq result (likelihood of being differentially expressed and False Discovery Rate). Fold Changes (CSS/MBHIB) and whether the Fold Changes are above or below the thresholds (< 0.4 or > 2.5) are provided.

Submission date

Dec 19, 2014

Last update date

May 15, 2019

Contact name

Silin Tang

E-mail(s)

[email protected]

Organization name

Cornell University

Department

Food Science