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Sample GSM2559788

Query DataSets for GSM2559788

Status

Public on Mar 30, 2018

Title

Amp_treated_SK36 strain_T30_Rep1

Sample type

SRA

Source name

Ampicillin-treated SK36 after 30 min

Organism

Streptococcus sanguinis

Characteristics

strain: SK36
growth phase: late-log
treated with: 0.125 µg/ml of Ampicillin for 30min

Growth protocol

Untreated cells were incubated at 37°C under anaerobic conditions in brain-heart infusion media and harvested at late-log phase. Treated cells were exposed to sub-inhibitory Ampicillin dose at late-log phase for either 10, 20, or 30 minutes.

Extracted molecule

total RNA

Extraction protocol

Cells were lysed and RNA was extracted by FastPrep lysing matrix beads. Clean-up and DNase I treatment was conducted by QIAGEN RNeasy mini kit, according to the manufacterer's protocol. Illlumina RiboZero Magnetic Kit for bacteria was used to deplete ribosomal RNA from 2 µg of total RNA.
NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England BioLabs) was used for the RNA-seq library preparation according to the manufacturer’s protocol. Briefly, ribosomal-depleted RNA was fragmented followed by first-strand cDNA synthesis from random primers using ProtoScript II Reverse Transcriptase (New England BioLabs). Second strand cDNA was synthesized and purified. End repair was performed on the double-stranded cDNA and primed with the addition of 5’-phosphorylated dA-tailed ends using T4 DNA polymerase, Klenow DNA polymerase and T4 polynucleotide kinase (New England BioLabs). This was immediately followed by adaptor ligation (New England BioLabs) and purified. Samples were PCR-amplified for 12 cycles with Phusion HiFi polymerase (New England Biolabs) with paired-end primers and a randomly chosen unique barcode (NEB Next Multiplex Oligs for Illumina - Index Primers 2,3,4,5,6,8,10,11,12,13,15,16). Agencourt AMPure XP Beads (Beckman Coulter) were used for all purification steps.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

replicate 1

Data processing

Standard Illumina pipeline. Real Time Analysis (RTA v. 1.18.66.3) software used for image processing, intesity extraction and basecalling based on intensities. CHASTITY software used for quality filter of basecalling.
Reads obtained from RNA-sequencing were aligned against the Streptococcus sanguinis SK36 genome using using Rockhopper v. 2.03
Analyses were run on default parameter settings to obtain expression data of the treated samples compared to untreated SK36. Significance was represented by a q-value ≤ 0.01 adjusted for a false discovery rate of 1%.
Genome_build: Streptococcus sanguinis reference genome; NC_009009.1
Supplementary_files_format_and_content: tab-delimited text file includes transcription and translation start/stop site, strand direction, gene name and ID, gene product, raw and normalized counts for each replicate, Reads Per Kilobase of exon per Megabase of library size (RPKM), expression, p-value and Rockhopper's adjusted q-value for the mutant expression vs. the wild-type SK36 expression

Submission date

Mar 30, 2017

Last update date

May 15, 2019

Contact name

Fadi Elias El-Rami

E-mail(s)

[email protected]

Organization name

Virginia Commonwealth University

Department

Microbiology & Immunology