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Sample GSM1880696

Query DataSets for GSM1880696

Status

Public on Feb 11, 2016

Title

ΔBCHL-RH rep3

Sample type

SRA

Source name

BHI broth

Organism

Listeria monocytogenes 10403S

Characteristics

strain: 10403S::deltaBCHL Prha-sigH
growth stage: log phase
induction: Rhamnose addition

Treatment protocol

250 μl 1M rhamnose stock solution was added to 5 ml OD600 0.4-0.5 bacterial cultures (for a final concentration of 50 mM rhamnose), followed by incubation at 37°C for an additional 30 min.

Growth protocol

strains were streaked from frozen BHI stock, stored at -80°C in 15% glycerol, onto a BHI agar plate, followed by incubation at 37°C for 24 h. A single colony was subsequently inoculated into 5 ml of BHI broth in 16 mm tubes, followed by incubation at 37oC with shaking (230 rpm) for 18 h (Series 25 Incubator, New Brunswick Scientific, Edison, NJ). After 18 h, 50 μl BHI culture was inoculated into fresh 5 ml BHI broth and grown to OD600 0.4-0.5 at 37°C.

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 2500

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 10403S genome using the BWA-MEM with default settings.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 2.2.0 package available from Bioconductor. Genes were considered differentially expressed if they showed a false discovery rate (FDR) < 0.05 and a fold change (FC) ≥ 2.0 (meaning genes were upregulated on Prha-sigH) or FC ≤ 0.5 (meaning genes were downregulated on Prha-sigH).
Sliding window analysis: the 10403S genomes was divided into windows of 51 nt (window size) with 25 nt overlap (window sliding) and the RNA-Seq coverage was obtained for each of the 116,123 resulting windows. Bayseq version 2.2.0 was then used, as described above, to identify windows with significant differential expression between the ΔBCHL::Prha-sigH strain and the ΔBCHL::Prha strain. Fold change was also calculated as described above and windows presenting an FDR < 0.05 and FC > 2.0 were considered for further analysis. Windows matching the required thresholds were then sorted and overlapping windows were considered as being part of the same fragment. Fragments that were mapped to the same gene or operon were considered as being part of the same transcriptional unit (TU). The 5’ end of significant TUs were then manually scanned for promoter sequences, as described above.
Genome_build: 10403S genome (genebank accession number: NC_017544), assembly ASM16869v2
Supplementary_files_format_and_content: An excel spreadsheet (.xlsx) file: Raw count data and normalized count data (from BaySeq output) for each coding gene in 10403S. BaySeq result (likelihood of being differentially expressed and False Discovery Rate). Fold Changes (ΔBCHL-RH/ΔBCHL-R) and whether the Fold Changes are above or below the thresholds (< 0.5 or > 2.0) are provided. And a text (.txt) file containing the output from BaySeq with normalized coverage for each 51 nt window and Fold Changes (ΔBCHL-RH/ΔBCHL-R)

Submission date

Sep 14, 2015

Last update date

May 15, 2019

Contact name

Martin Wiedmann

E-mail(s)

[email protected]

Organization name

Cornell University

Department

Food Science