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| Status |
Public on May 05, 2014 |
| Title |
Ecoli_ΔgreB_NETseq |
| Sample type |
SRA |
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| Source name |
Bacteria
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| Organism |
Escherichia coli |
| Characteristics |
strain background: MG1655
genotype/variation: ΔgreB
molecule subtype: Nascent RNA
media: MOPS EZ Rich Defined Media
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| Growth protocol |
For each sample, 500 mL liquid cultures were grown in 2.8 L flasks with shaking (180 rpm) at 37 °C from an OD (600 nm) 0.05 to OD 0.45 ± 0.05. Cells were grown in MOPS EZ rich defined medium with 0.2% glucose (Teknova).
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cell culture was rapidly filtered in 250 mL increments at 37 °C over 0.22 μm nitrocellulose filters (GE) and frozen in liquid nitrogen to simultaneously halt all transcriptional progress. Frozen cells (100 μg) were pulverized on a Qiagen TissueLyser II mixer mill 6 times at 15 Hz for 3 min in the presence of 500 μL frozen lysis buffer (20 mM Tris pH 8, 0.4% Triton X-100, 0.1% NP-40, 100 mM NH4Cl, 50 U/mL SUPERase•In (Ambion)) and 1X protease inhibitor cocktail (Complete, EDTA-free, Roche), supplemented with 10 mM MnCl2. The lysate was resuspended on ice by pipetting. RQ1 DNase I (110 U total, Promega) was added and incubated for 20 min on ice. The reaction was quenched with EDTA (25 mM final), which releases polysomes from the transcript and reduces contamination from ribosomal RNA and ribosome-associated tRNAs. The lysate was clarified at 4 °C by centrifugation at 20,000 g for 10 min. The lysate was loaded onto a PD MiniTrap G-25 column (GE Healthcare) and eluted with lysis buffer supplemented with 1 mM EDTA. For nascent RNA purification, the clarified lysate was added to 0.5 mL anti-FLAG M2 affinity gel (Sigma Aldrich). The affinity gel was washed twice with lysis buffer supplemented with 1 mM EDTA before incubation with the clarified lysate at 4 °C for 2.5 h with nutation. The immunoprecipitation was washed 4 × 10 mL with lysis buffer supplemented with 300 mM KCl, and bound RNAP was eluted twice with lysis buffer supplemented with 1 mM EDTA and 2 mg/mL 3XFLAG peptide (Sigma Aldrich). Nascent RNA was purified from the eluate using the miRNeasy kit (Qiagen).
The full library construction protocol was described in detail previously (Churchman et al., Nature 2011). Purified nascent RNA was ligated to a 5' adenylated DNA oligo and fragmented to generate RNA ranging from 30-100 nt. The RNA was reverse transcribed, and the single-stranded DNA circularized and PCR amplified.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
library strategy: NET-seq
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| Data processing |
Basecalls performed using Casava versions 1.6 or 1.7.
Sequenced reads were trimmed for adaptor sequence.
Trimmed reads were aligned using Bowtie v0.12.7 against the reference genome using parameters -a -v 3 -m 1
Bowtie alignments against the reference genome were converted to wiggle files. The position of each alignment was mapped to the 3' end of the nascent transcript.
Genome_build: E. coli: NC_000913
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).
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| Submission date |
Apr 10, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Matthew H Larson |
| Organization name |
Grail, Inc.
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| Street address |
499 Illinois St
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| City |
San Francisco |
| State/province |
CA |
| ZIP/Postal code |
94158 |
| Country |
USA |
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| Platform ID |
GPL14548 |
| Series (1) |
| GSE56720 |
Nascent elongating transcript sequencing (NET-seq) for Escherichia coli and Bacillus subtilis reveals a consensus pause sequence enriched at translation start sites. |
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| Relations |
| BioSample |
SAMN02725506 |
| SRA |
SRX515176 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1367306_Ecoli_greB.minus.wig.gz |
7.0 Mb |
(ftp)(http) |
WIG |
| GSM1367306_Ecoli_greB.plus.wig.gz |
6.9 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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