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Sample GSM1300282

Query DataSets for GSM1300282

Status

Public on Apr 24, 2014

Title

mRNA-seq in rich defined media

Sample type

SRA

Source name

bacteria

Organism

Escherichia coli

Characteristics

strain: MG1655
media: fully supplemented MOPS glucose media

Growth protocol

All cultures were based on MOPS media with 0.2% glucose (Teknova), with either full supplement (Neidhardt et al., 1974). An overnight liquid culture was diluted 400-fold into 200 ml fresh media. The culture was kept in a 2.8-liter flask at 37C with aeration (180 rpm) until OD600 reached 0.3.

Extracted molecule

total RNA

Extraction protocol

Extraction was performed as described in detail previously (Li et al., 2012; Oh et al., 2011). 200 ml of cell culture was rapidly filtered at 37C by passing through a nitrocellulose filter. Cell pellets was were rapidly collected using a pre-warmed metal table crumber, flash frozen in liquid nitrogen, and combined with frozen droplets of lysis buffer. Cells and lysis buffer were pulverized in 10 ml canisters (Retsch) pre-chilled in liquid nitrogen using Qiagen TissueLyser II. Pulverized lysate was thawed on ice and clarified by centrifugation at 4C.
Ribosome protected mRNA fragments were size selected via gel purification, and ligated to 5' adenylated DNA oligo. After reverse transcription, the single stranded DNA was circularized, and PCR amplified [Oh et al,. Cell 147, 1295 (2011)]. More at G.W. Li, D. Burkhardt, C.A. Gross, J. S. Weissman (Cell).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

mRNA
mrna-rdm-pooled_f.wig
mrna-rdm-pooled_r.wig

Data processing

Basecalls performed using Casava versions 1.6 or 1.7.
Sequenced reads were trimmed for adaptor sequence.
Trimmed reads were sequentially aligned using Bowtie v0.12.7 to E coli rRNA and noncoding RNA allowing one mismatch. Reads aligning to any of these indices were discarded.
The remaining reads were aligned using Bowtie v0.12.7 against E. coli MG1655 genome using parameters -v1 -m2 -k1.
Bowtie alignments against the E coli genome were converted to wiggle files. The position of each alignment is distributed into several nucleotides in the center of the footprint. For each footprint read, the center residues that are at least 10 nucleotides away from either ends were given the same score, which is weighted by the length of the fragment [Oh et al,. Cell 147, 1295 (2011)]. Scores therefore represent the number of read alignments attributed to each genomic position under each scoring scheme.
Genome_build: NC000913.2
Supplementary_files_format_and_content: wiggle files with two columns: first column containing chromosome positions and second column containing the number of reads mapped to the position (see publication for details).

Submission date

Jan 02, 2014

Last update date

May 15, 2019

Contact name

Gene-Wei Li

E-mail(s)

gene-wei.li@ucsf.edu

Organization name

UCSF

Street address

1700 4th Street, BH404