|
| Status |
Public on Jul 16, 2015 |
| Title |
pBAD-MS2 |
| Sample type |
SRA |
| |
|
| Source name |
Bacterium
|
| Organism |
Escherichia coli str. K-12 substr. MG1655 |
| Characteristics |
genotype/variation: rne131
growth phase: exponential and stationary phases
|
| Treatment protocol |
0.1% arabinose was added to induce the expression of MS2 aptamer (fused to RyhB terminator) during 10 min. Cells were chilled for 10 min on ice. Cells were then centrifuged, resuspended in 1 mL of buffer A (20 mMTris-HCl at pH 8.0, 150 mM KCl, 1 mM MgCl2, 1 mM DTT), and centrifuged again.
|
| Growth protocol |
Cells were harvested in exponential (OD600nm=0.5; 100 mL LB medium) and in stationary phase (OD600nm=1; 100 mL LB medium).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were resuspended in 3 mL of buffer A and lysed using a French Press (430 psi, four times). Lysate was then cleared by centrifugation. The soluble fraction was subjected to affinity chromatography. The column was prepared by adding 100 µL of amylose resin (New England Biolabs) to Bio-Spin disposable chromatography columns (Bio-Rad). The column was washed with 3 mL of buffer A. Next, 200 pmol of MS2-MPB protein was immobilized on the amylose resin, and the column was washed with 2 mL of buffer A. The cleared lysate was then loaded onto the column, which was washed with 8 mL of buffer A. RNA was eluted from the column with 1 mL of buffer A containing 15 mM maltose. Eluted RNA was extracted with phenol-chloroform, followed by ethanol (3 vol) precipitation of the aqueous phase in the presence of 20 mg of glycogen. RNA samples were treated with TURBO DNA-free (DNase treatment) and precipitated again.
cDNA libraries were prepared with ScriptSeq™ v3 RNA-Seq Library Preparation Kit (Epicentre)
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina MiSeq |
| |
|
| Data processing |
Basecalls performed using MCS Version 2.4.1.3
Reads were aligned to the Escherichia coli K12 genome assembly using Bowtie for Illumina (Galaxy Project)
Read counts were normalized by coverage according to Oshlack et al., 2010 (i.e. divided by total number of proof reads for each sample)
Genome_build: Escherichia coli K12
Supplementary_files_format_and_content: tab-delimited text file include normalized reads for each sample and pBAD-MS2/pNM12 ratio
|
| |
|
| Submission date |
Apr 06, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Eric Massé |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Sherbrooke
|
| Department |
Biochemistry
|
| Lab |
Eric Massé Laboratory
|
| Street address |
3201, Jean Mignault
|
| City |
SHERBROOKE |
| State/province |
Québec |
| ZIP/Postal code |
J1E 4K8 |
| Country |
Canada |
| |
|
| Platform ID |
GPL17439 |
| Series (2) |
| GSE67606 |
Identification of RNAs bound to MS2 aptamer using MS2-affinity purification coupled with RNA sequencing (MAPS) |
| GSE67607 |
DsrA regulatory RNA represses both hns and rbsD mRNAs through distinct mechanisms in Escherichia coli |
|
| Relations |
| SRA |
SRX978919 |
| BioSample |
SAMN03461668 |
