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Sample GSM1130903

Query DataSets for GSM1130903

Status

Public on Aug 01, 2013

Title

error-prone in vitro (Mn2+)

Sample type

SRA

Source name

error-prone in vitro MnCl2

Organism

Escherichia coli

Characteristics

strain: MG1655
mrna synthesis: in vitro MnCl2

Growth protocol

Cells were cultured in LB medium containing ampicillin at 28˚C. The overnight cell culture was inoculated into the fresh medium at 1/70 (v/v) and was incubated for 2 hr at 28˚C (OD600 reached 0.35) and then for 2 hr at 42˚C (OD600 reached 2.3) to induce the PR promoter.

Extracted molecule

total RNA

Extraction protocol

In vitro RNA preparation: The 5.7 kb RNA was purified from the digested DNA, NTPs, abortive oligo-RNA products, and proteins by Acidic phenol extraction, G50 spin column, followed by EtOH precipitation.
In vivo RNA preparation: The cells in 200 ml culture were harvested and resuspended with a solution containing 0.5% SDS, 20 mM sodium acetate (pH 5.5), and 10 mM EDTA. The suspended cells were mixed with an equal volume of pre-warmed saturated phenol (20 mM sodium acetate, 10 mM EDTA pH 5.5) and incubated for 5 min at 60 C. The mixture was centrifuged, and RNA and DNA were precipitated with ethanol from the supernatant. The pellet was dissolved in DNase I buffer with 10U of DNaseI and incubated for 30 min. RNA was separated from the digested DNA by acidic phenol extraction followed by G-50 Micro column (GE Healthcare) purification, and then precipitated with ethanol. The pellet was dissolved in diethylpyrocarbonate-treated water and used for cDNA synthesis.
The cells culture were harvested and resuspended with a solution containing 0.5% SDS, 20 mM sodium acetate (pH 5.5), and 10 mM EDTA. The suspended cells were mixed with an equal volume of pre-warmed saturated phenol (20 mM sodium acetate, 10 mM EDTA pH 5.5) and incubated for 5 min at 60˚C.
We established a method for preparing five different cDNA libraries each with its own barcode for Illumina sequencing. Each 6-nt barcode allows multiplexing all five in vitro and in vivo preparations in a single sequencing analysis. our method introduces internal control sequences to the library that are subjected to the artifact errors, but are not for RNAP errors. The 5’ fragment of the 5.7 kb RNA transcripts was reverse transcribed to make the cDNA. The cDNA was subjected to PCR reactions that generated six 200 bp segments. The primers contained a specific barcode for each of the five starting preparations and the inner Illuminasequencing adapters. The 2nd-step of PCR generated the final cDNA libraries for the Illumina sequencing by using the 1st-step PCR product as a template and primers containing the outer sequencing adapters in the 5’ tails.
mRNA-seq with barcode (Illumina TruSeq Index 1-5)

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

mRNA synthesized in vitro transcription

Data processing

CASAVA version 1.4
A single large fastq format file of high quality reads (Q ≥ 30) was split into about 10 smaller files by using a shell script splitReads.sh
The obtained reads were aligned and mapped to the pPR9 plasmid DNA sequences using Bowtie 0.12.7.
The numbers of 4 bases A, T, G, C, and N were counted in each position of the mapped reads by using the program SAMtools 0.1.18 with supplemental use of a Perl script.
Each type of error rates per position was determined as the number of sequence reads with a particular type of base-substitution divided by the number of the reads with the reference base in each DNA position.
Genome_build: pPR9 plasmid (ref is Kashlec et al., 1989, PMID: 2547695)
Supplementary_files_format_and_content: tab-delimited text file of transition error rate per position. The position is corresponding to the position of "Sequenced region of pPR9 plasmid" (one of the attached txt file).

Submission date

Apr 29, 2013

Last update date

May 15, 2019

Contact name

Masahiko Imashimizu

E-mail(s)

imashimizum@mail.nih.gov

Organization name

NCI

Department

NCI/CCR