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Sample GSM1086991

Query DataSets for GSM1086991

Status

Public on Apr 23, 2013

Title

induced 1

Sample type

SRA

Source name

bacterial colonies

Organism

Bartonella henselae

Characteristics

strain: MQB307
treatment: 500 uM IPTG

Growth protocol

MQB307 was grown on Columbia agar plates containing 5% defibrinated sheep blood (CBA plates) supplemented with 30 mg/l kanamycin with (induced condition) or without (uninduced condition) 500 uM isopropyl β-D-thiogalactoside (IPTG) at 35°C and 5% CO2 for 60 h

Extracted molecule

total RNA

Extraction protocol

RNA was isolated from bacterial cells using a modified hot-phenol extraction, followed by DNase I digestion, RNA cleanup (RNeasy Mini Kit, Qiagen). Integrity of total RNA was verified on an Agilent 2100 Bioanalyser using the RNA 6000 Nano LabChip kit (Agilent Technologies). The purified RNA was used directly for downstream application or stored at -70°C after precipitation with 2.5 volumes ethanol and 1/10 volume 3 M sodium acetate, pH 5.2.
The Whole Transcriptome library was produced using the Ribominus kit, Bacteria Module (Invitrogen PN 45-7014) and the SOLiD™ Total RNA-seq kit (Applied Biosystems p/n 4445374). Briefly, 5 μg of total RNA was depleted of rRNA and then fragmented using RNase III. Ligation of the adaptor mix and reverse transcription were performed following the manufacturer's protocol. cDNA libraries were size selected for fragments between 150 and 250 bp, amplified for 18 cycles of PCR using barcoded adaptor primers and purified with the PureLink PCR micro kit (Invitrogen PN K310050). Library size and concentrations were assessed on a Bioanalyzer (Agilent) and on a Qubit fluorometer (Invitrogen), respectively. The whole transcriptome library was used for emulsion-PCR based on a concentration of 0.5 pM. Sequencing beads from four barcoded libraries (2 biological replicates for the uninduced and induced condition, respectively) were pooled together and loaded on a full SOLiD™-4 slide (Applied Biosystems), according to manufacturer's instructions. SOLiD™ ToP Sequencing F3-Tag MM50 chemistry was used to produce 50 base sequencing reads.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

AB SOLiD 4 System

Data processing

The sequenced reads were mapped using the BioScope 1.3.1 mapping pipeline (mapreads software using local alignment strategy).
SAM files were subsequently further processed to remove among all uniquely mapping reads those with an average mapping/read quality below 20, or more than two mismatches.
For the class of doubly mapped reads, we eliminated reads from duplicated 16S and 23S rRNA genes (accounting for >99%) and then filtered instances where, based on edit distance and map quality as selection criteria, one of the two mappings exhibited a better match with the reference genome sequence. This was repeated for triply mapped reads (including the 5S rRNA genes) and up to 4-7 mapped genome positions per read.
The count data summary for annotated B. henselae ORFs was generated using the HTSeq package, the quality of the reads was visually inspected with the HTSeq-qa program.
Transcript abundance was estimated via RPKM values. The number of mapped and filtered reads for each B. henselae protein-coding gene was divided by the length of the gene (in kilobases) and the number of mapped and filtered reads to all B. henselae protein-coding genes in the experiment (in million reads).
Differential transcript expression analysis was carried out with the R package DESeq (version 1.6.1).
Genome_build: B. henselae Houston-1 strain (NCBI RefSeq acc. NC_005956.1, slightly modified to include the bepD-gfpmut2 chromosomal reporter fusion, and retain the deleted batR-batS sequence)
Genome_build: ASM4670v1
Supplementary_files_format_and_content: Tab-delimited text file includes for each protein-coding gene reads and RPKM values per sample, as well as differential expression analysis for replicate 2.

Submission date

Feb 22, 2013

Last update date

May 15, 2019

Contact name

Ulrich Omasits

E-mail(s)

omasitsu@ethz.ch

Organization name

ETH Zurich

Department

IMSB Institute of Molecular Systems Biology