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| Status |
Public on Aug 27, 2016 |
| Title |
Gradient fraction 05 |
| Sample type |
SRA |
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| Source name |
bacterial cells
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| Organism |
Salmonella enterica subsp. enterica serovar Typhimurium |
| Characteristics |
strain: SL1344
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| Treatment protocol |
For lysis, ~200 OD600 of bacterial culture were harvested by centrifugation at 4000 g at 4°C for 15 min, washed thrice with ice-cold 1×TBS and resuspended in 500 µl of the lysis buffer (20 mM Tris-HCl, pH7.5, 150 mM KCl, 1 mM MgCl2, 1 mM DTT, 1 mM PMSF, 0.2% Triton X100, 20 U/ml DNase I, Thermo Scientific, 200 U/ml SUPERase-IN, Life Technologies). Lysis was carried out on a Retsch MM400 machine at 30 Hz for 10 min in the presence of 750 µl 0.1 mm glass beads. The lysate was cleared by centrifugation at 14,000 g at 4°C for 10 min and layered on a linear 10%-40% (w/v) glycerol gradient in the same buffer without DNase I nor SUPERase-IN, formed in a Beckman SW40Ti tube. The gradient was centrifuged at 100,000 g (23,700 rpm) for 17 h at 4°C and fractionated in 20 equal fractions by pipetting. OD260 for each fraction was measured with Nanodrop.
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| Growth protocol |
LB, 37°C, 220 rpm, OD600=2.0
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| Extracted molecule |
total RNA |
| Extraction protocol |
Each fraction was deproteinized by addition of 1% SDS and 1 volume of hot phenol and shaking at 1,500 rpm at 55°C for 5 min. Phases were separated by centrifugation at 12,000 g for 15 min at 4°C. The aqueous phases were added glycogen to 50 µg/ml and RNA was precipitated with 60% isopropanol. The RNA pellets were dissolved in 35 µl of DEPC-treated sterile MilliQ water and stored at -80°C. Each fraction sample was added 85 pg/µl of the spike-in RNA (5’P-CUCGUCCGACGUCACCUAGA, IBA).
RNA-seq libraries were prepared by Vertis AG (Freising-Weihenstephan, Germany). Briefly, RNA was polyadenylated with poly(A) polymerase, 5’-triphosphates were removed with tobacco acid pyrophosphatase followed by ligation of a 5’-adapter. First-strand cDNA synthesis was performed with the use of an oligo(dT) barcoded adapter primer and the M-MVL reverse transcriptase. The resulting cDNA was PCR-amplified with a high fidelity DNA polymerase. cDNA was purified with the Agencourt AMPure XP kit (Beckman Coulter Genomics).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Demultiplexing
Fastq quality trimming using a cut-off value of 20 (via READemption)
Fastq to fasta conversion (via READemption)
Size filtering: discarding reads shorter than 12 nt (via READemption)
Read mapping using segemehl (via READemption)
Coverage calculation and normalisation (via READemption)
Genome_build: NC_016810.1, NC_017718.1, NC_017719.1, NC_017720.1
Supplementary_files_format_and_content: wiggle
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| Submission date |
Nov 04, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Konrad U. Förstner |
| E-mail(s) |
foerstner@zbmed.de
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| Organization name |
ZB MED - Information Centre for Life Sciences
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| Department |
Information Services
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| Lab |
Förstner Lab
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| Street address |
Gleueler Str. 60
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| City |
Cologne |
| State/province |
North Rhine-Westphalia |
| ZIP/Postal code |
50931 |
| Country |
Germany |
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| Platform ID |
GPL17070 |
| Series (2) |
| GSE62986 |
Fraction-wise RNA-seq of the Salmonella Typhimurium SL1344 cell lysate resolved on a 10-40% glycerol gradient |
| GSE62988 |
Partitioning of a RNA interactome by gradient profiling (Grad-seq) |
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| Relations |
| BioSample |
SAMN03161919 |
| SRA |
SRX751158 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1537628_Fraction_05_div_by_4944091.0_multi_by_3249290.0_forward.wig.bz2 |
4.4 Mb |
(ftp)(http) |
WIG |
| GSM1537628_Fraction_05_div_by_4944091.0_multi_by_3249290.0_reverse.wig.bz2 |
4.6 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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