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Sample GSM2978395 Query DataSets for GSM2978395
Status Public on Mar 25, 2019
Title PDD_P2_78
Sample type SRA
 
Source name Trimethoprim
Organism Escherichia coli BW25113
Characteristics strain: BW25113
type: Gram-negative bacteria
moa: Folic Acid synthesis inhibitor
phenotype: EC90 of phenotype
treatment time: ~ 25 min
treatment concentration: 50 uM
Treatment protocol bacteria were treated with different antibiotics for ~ 25 min till ~OD 0.2 in 2 ml tubes
Growth protocol bacteria were grown in iso-sensitest medium
Extracted molecule total RNA
Extraction protocol after treament bacteria were resuspended in QiaGen RNAprotect Bacteria Reagent (QiaGen #76506), incubated for 5min, centrifuged, and flash frozen on dry ice. Total RNA was extracted by incubating bacteria in Enzymatic Lysis Buffer (lysozyme & proteinase K) for 5 min followed by addition of QiaGen RLT Lysis Buffer and RNA purification using the QiaGen RNeasy Mini kit combined with DNase treatment on a solid support (QiaGen #74104). RNA quality assessment and quantification was performed using microfluidic chip analysis on an Agilent 2100 bioanalyzer (Agilent Technologies).
For RNA-sequencing library preparation, 1000 ng total RNA was used as input. First, bacterial ribosomal RNA was depleted using the Ribo-Zero Magnetic Kit Bacteria (Illumina #MRZB12424). After depletion, RNA was resuspended in TruSeq Total RNA Sample Prep Kit Fragmentation buffer (8.5 ul RNA and 8.5 buffer) and reversed transcribed into cDNA using random hexamer primer. Then cDNA was further processed for the construction of sequencing libraries according to the manufacturer's recommendations using the TruSeq Stranded mRNA Sample Prep Kit (Illimina #RS-122-2101). Sequencing was performed with the Illumina TruSeq SBS Kit v4-HS chemistry (Illumina #FC-401-4003) on an Illumina HiSeq2500 instrument with 50 cycles of 2x50 bp paired-end sequencing.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2500
 
Data processing Illumina CASAVA v1.8.2 software used for basecalling and fastq file generation
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to Escherichia coli str. K-12 substr. MG1655, complete genome (GenBank: U00096) genome using bowtie2
Reads Per Kilobase of exon per Megabase of library size (RPKM) were calculated using a protocol from Chepelev et al., Nucleic Acids Research, 2009. In short, exons from all isoforms of a gene were merged to create one meta-transcript. The number of reads falling in the exons of this meta-transcript were counted and normalized by the size of the meta-transcript and by the size of the library.
Genome_build: Escherichia coli str. K-12 substr. MG1655, complete genome (GenBank: U00096)
Supplementary_files_format_and_content: tab-delimited text files in GCT format include read counts of uniquely and fraction of multiple mapped reads (counts.gct.gz), and normalized counts RPKM (rpkms.gct.gz) values for each sample
 
Submission date Feb 05, 2018
Last update date Mar 25, 2019
Contact name Roland Schmucki
E-mail(s) roland.schmucki@roche.com
Organization name F. Hoffmann - La Roche AG
Street address Grenzacherstrase
City Basel
ZIP/Postal code 4058
Country Switzerland
 
Platform ID GPL20227
Series (1)
GSE110137 Mode of action characterization of antibiotics using expression profile fingerprints generated by RNA-sequencing technology
Relations
BioSample SAMN08466806
SRA SRX3648483

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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