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Sample GSM1310004

Query DataSets for GSM1310004

Status

Public on Apr 10, 2014

Title

EC_Cont2_RNA

Sample type

SRA

Source name

Untreated E. coli cells

Organism

Escherichia coli

Characteristics

strain: JM83
genotype/variation: wild type
treated with: none (untreated control)
molecule subtype: total RNA, rRNA depleted

Growth protocol

Overnight cultures of strain JM83 were diluted 1/100 in LB medium and grown in microtiter plates. To study the effects of trimethoprim, cells grown without antibiotics were compared to cells grown with 0.5 μg/ml trimethoprim.

Extracted molecule

total RNA

Extraction protocol

For all the samples; when 1/3 of the maximum OD600 was reached, cells were harvested by centrifugation (7,500 rcf for 5 min) and frozen. For RNA isolation, cells were lysed by bead beating and RNA was purified using phenol-chloroform extractions and ethanol precipitations. DNA was removed from the sample with RNase-free DNase I (Fermentas) treatment for 45 min. Ribolock (Fermentas) was added to avoid RNA degradation.
Library preparation was performed by vertis Biotechnologie AG, according to the following protocol: RNA samples were first treated with rDNase. From the total RNA samples, ribosomal RNA molecules were depleted using the Ribo-Zero rRNA Removal Kit (Bacteria, Epicentre). the rRNA depleted RNA samples were fragmented with ultrasound (2 pulses of 30 sec at 4°C). Firststrand cDNA synthesis was primed with a N6 randomized primer. Then, Illumina TruSeq sequencing adapters were ligated to the 5' and 3' ends of the cDNA. The cDNA was finally amplified with PCR (16-18 cycles, depending on sample) using a proof reading enzyme. Aliquots of each library were analyzed by capillary electrophoresis.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

processed data file: EC_Cont_RNA.txt

Data processing

Illumina Casava 1.8 software was used for basecalling.
S. pneumoniae reads were mapped to the D39 whole genome with Rockhopper version 1.21, using default parameters. E. coli reads were mapped to the K-12, substr. MG1655 whole genome with Bowtie 2, using parameters --mixed --discordant -D 10 -R 2 -N 0 -L 22 -i S,0,2.50.
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. For the S. pneumoniae samples this was done internally by Rockhopper version 1.21 after aligning reads.
Genome_build: Streptococcus pneumoniae D39 (assembly ASM1436v1)
Genome_build: Escherichia coli K-12, substr. MG1655 (assembly ASM584v2)
Supplementary_files_format_and_content: tab-delimited text files include RPKM values for each Sample.

Submission date

Jan 17, 2014

Last update date

May 15, 2019

Contact name

Jelle Slager

E-mail(s)

j.slager@rug.nl

Organization name

University of Groningen

Department

Molecular Genetics