GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM3450052

Query DataSets for GSM3450052

Status

Public on Oct 30, 2018

Title

E. coli-WT-untreated-mRNA-rep2

Sample type

SRA

Source name

bacteria

Organism

Escherichia coli

Characteristics

strain: BW25113
nabh4 treatment: none

Extracted molecule

total RNA

Extraction protocol

Hot acid phenol extraction
RNA-seq: Methods in molecular biology, 1038, 213-231 (2013) with RNA fragmentation step included. Small RNA-seq: NEBNext Multiplex Small RNA Kit.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Description

Libraries were prepared from total RNA. Briefly, after NaBH4/mock treatment the RNA was reverse transcribed to cDNA using a random primer with Illumina overhang, and an adapter was ligated to the 3'-end of the cDNA. The DNA was amplified with PCR using primers compatible with Illumina sequencing.
coli_mrna_rrna_out.txt

Data processing

Sequencing reads were processed with Cutadapt (version 1.14) to remove adapter sequences using the setting -a AGATCGGAAGAGCACACGTCT. For reads generated nextseq platform, the option --nextseq-trim=20” was included.
All samples (except small RNA-Seq experiments): The RNAProbR preprocessing.sh script (http://people.binf.ku.dk/~jvinther/data/rna_probing/) was used to remove the 7 base barcode from the sequencing read using the options “-b NNNNNNN -t 9”
Reads were aligned with bowtie2 (version 2.2.3) using high sensitivity options “--local -N 1 -D 20 -R 3 -L 15”
All samples (except small RNA-Seq experiments): Potential PCR duplicates among the sequencing reads were removed by discarding all but one of the reads having identical barcode and mapping to the same position using the collapse.sh script (available at http://people.binf.ku.dk/jvinther/data/RNA-seq).
The mapped reads for the control and NaBH4 treated samples were processed with the mpileup tool from the samtools package (version 1.7).
Genome_build: E. coli strain bw25113 (ensembl cDNA release 87), S. cerevisiae (ensembl cDNA release 84). rRNA sequences were obtained were obtained from the CRW database (http://www.rna.ccbb.utexas.edu). tRNA sequences were obtained from the genomic tRNA database (http://gtrnadb.ucsc.edu/index.html).
Supplementary_files_format_and_content: The mutation frequency file is the output from the GetFreq function (available at https://github.com/jeppevinther/m7g_map_seq) includes a number of statistics for each position, including coverage, mutation frequency for the treated and control samples and the mutation rate difference between the treated and control samples. It includes p-values for the observed mutation rates being different in the treated and the control samples and for the replicate samples being different.

Submission date

Oct 29, 2018

Last update date

Oct 30, 2018

Contact name

Jeppe Vinther

Organization name

University of Copenhagen

Department

Biology

Lab

Vinther Lab