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Status |
Public on Mar 25, 2019 |
Title |
PDD_P2_31 |
Sample type |
SRA |
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Source name |
Chloramphenicol
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Organism |
Escherichia coli BW25113 |
Characteristics |
strain: BW25113 type: Gram-negative bacteria moa: Protein synthesis phenotype: EC90 of phenotype treatment time: ~ 25 min treatment concentration: 25 uM
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Treatment protocol |
bacteria were treated with different antibiotics for ~ 25 min till ~OD 0.2 in 2 ml tubes
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Growth protocol |
bacteria were grown in iso-sensitest medium
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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.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 2500 |
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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
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Submission date |
Feb 05, 2018 |
Last update date |
Mar 25, 2019 |
Contact name |
Roland Schmucki |
E-mail(s) |
roland.schmucki@roche.com
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Organization name |
F. Hoffmann - La Roche AG
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Street address |
Grenzacherstrase
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City |
Basel |
ZIP/Postal code |
4058 |
Country |
Switzerland |
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Platform ID |
GPL20227 |
Series (1) |
GSE110137 |
Mode of action characterization of antibiotics using expression profile fingerprints generated by RNA-sequencing technology |
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Relations |
BioSample |
SAMN08466794 |
SRA |
SRX3648437 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
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