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| Status |
Public on Mar 21, 2017 |
| Title |
ID-003340-02_Hfq_FLAG |
| Sample type |
SRA |
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| Source name |
Hfq FLAG tag, OD 0.5
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| Organism |
Neisseria meningitidis 8013 |
| Characteristics |
sample type: anti-FLAG co-IPed
strain: 8013
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| Growth protocol |
N.meningitidis 8013 and its derivatives were either grown on blood agar plates (bioMerieux) or on GC medium base (GCB) plates (Difco) containing Kellogg’s supplements I and II (22.2 mM glucose, 0.68 mM glutamine, 0.45 mM co-carboxylase, 1.23 mM Fe(NO3)3; all from Sigma) and appropriate antibiotics. For liquid cultures, Neisseria grown on plates overnight were harvested and a starter culture was inoculated to a final OD600 nm of 1.0 in 5 ml GC medium base (GCB) liquid supplemented with Kellogg’s I and II (GCBL++). After one hour the starter culture was used to inoculate GCBL++ media to a final OD600 of 0.1. Bacteria were always grown at 37 °C at 220 rpm without added CO2 to the desired OD.
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| Extracted molecule |
total RNA |
| Extraction protocol |
For dRNA-seq: N. meningitidis 8013 cells were grown in 50 ml tubes at 37 °C under vigorous shaking in GCBL++ medium and samples were collected by centrifugation at OD600 of 0.5 and 1.5, corresponding to the mid logarithmic and late logarithmic/early stationary growth phase, respectively. Transcription and translation were terminated by the addition of STOP Mix [95% (vol/vol) EtOH and 5% (vol/vol) phenol] and cells were frozen in liquid nitrogen. Samples were stored at −80 °C until RNA preparation. Frozen cell pellets were thawed on ice, and resuspended in a lysis solution consisting of 800 µl of 0.5 mg/ml lysozyme in TE buffer (pH 8.0) and 80 µl 10% SDS. Bacterial cells were lysed by placing the samples for 1–2 minutes at 65°C in a water bath. Total RNA was extracted from the lysates using the hot phenol method (Blomberg 1990). For RIP-seq:For each strain cells equivalent to an OD600 of 50 were collected in parallel and subjected to Hfq coIP and control coIP as described previously (Dugar et al. 2016). Cells were harvested by centrifugation at 6,000g for 20 min at 4 °C. Afterwards, cell pellets were resuspended in 1ml Lysis Buffer (20mM Tris-HCl, pH 8.0, 150mM KCl, 1mM MgCl2, 1mM dithiothreitol (DTT)) and subsequently centrifuged (5 min, 11,000g,4 °C). The pellets were shock-frozen in liquid nitrogen and stored at -80 °C. Frozen pellets were thawed on ice and resuspended in 0.8 ml Lysis Buffer. An equal volume of glass beads was then added to the cell suspension. Cells were then lysed using a Retsch MM40 ball mill (30 s-1, 10min) in pre-cooled blocks (4 °C) and centrifuged for 2min at 15,200g, 4 °C. The supernatant was transferred to a new tube, and an additional 0.4 ml of Lysis Buffer was added to the remaining un-lysed cells with beads. Lysis of the remaining cells was achieved by a second round of lysis at 30 s-1 for 5min. Centrifugation was repeated and this second supernatant was combined with the first one. The combined supernatant was centrifuged again for 30 min at 15,200g, 4 °C for clarification and the resulting supernatant (lysate fraction) was transferred to a new tube. The lysate was incubated with 35 ml anti-FLAG antibody (Monoclonal ANTI-FLAG M2, Sigma, #F1804) for 30 min at 4 °C on a rocker. Next, 75 ml of Protein A-Sepharose (Sigma, #P6649), prewashed with Buffer A, was added and the mixture was rocked for another 30 min at 4 °C. After centrifugation at 15,200g for 1min, the supernatant was removed. Pelleted beads were washed five times with 1 ml Lysis Buffer. Finally, 500 ml Lysis Buffer was added to the beads and RNA and proteins were separated by phenol-chloroform-isoamyl alcohol (ROTH 985.1) extraction and precipitated as described previously (Chao et al. 2012).
For dRNA-seq: cDNA libraries of dRNAseq samples for were constructed by vertis Biotechnology AG, Germany (http://www.vertis-biotech.com/), as described previously for eukaryotic microRNA (Berezikov et al. 2006), but omitting the RNA size-fractionation step prior to cDNA synthesis. In brief, total RNA samples were used for the preparation of two libraries each, either covering all transcripts or being specifically enriched for primary transcripts by treatment with terminator exonuclease (TEX, Epicentre). Next, equal amounts of RNA samples were poly(A)-tailed with poly(A) polymerase. The 5'-triphosphate structures were removed with tobacco acid pyrophosphatase (TAP). Afterwards, a 5’-RNA adapter was ligated and the first-strand cDNA was synthesized with an oligo(dT)-adapter primer and MMLV reverse transcriptase. A PCR-based amplification step with a high-fidelity DNA polymerase was then used to increase the cDNA concentration to 20-30 ng/µl. A library-specific barcode for multiplexing was included in the 3'-sequencing adapter. For RIP-seq: RIP-seq samples were constructed according to instructions of the NEBnext Multiplex Small RNA library set for Illumina ECL but omitting the TEX treatment.
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| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina MiSeq |
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| Description |
anti-FLAG co-IPed RNA
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| Data processing |
Reads were trimmed for a Phred quality of 20 using the FASTX toolkit version 0.0.13
Adapter trimming using Cutadapt version 1.3
Reads were mapped using the READemption pipeline (Förstner et al., 2014), segemehl, and the lack remapper
Coverage plots (wig files) were produced by READemption
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| Submission date |
Aug 05, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Lars Barquist |
| E-mail(s) |
[email protected]
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| Organization name |
Helmholtz Institute for RNA-based Infection Research
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| Street address |
Josef-Schneider-Straße 2 / Bau D15
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| City |
Würzburg |
| State/province |
Bayern |
| ZIP/Postal code |
97080 |
| Country |
Germany |
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| Platform ID |
GPL22296 |
| Series (1) |
| GSE85252 |
Transcriptome mapping and Hfq RIP-seq of Neisseria meningitidis |
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| Relations |
| BioSample |
SAMN05514350 |
| SRA |
SRX2005113 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2263202_02_Hfq_FLAG_div_by_425675.0_multi_by_1000000.0_forward.wig.gz |
1.1 Mb |
(ftp)(http) |
WIG |
| GSM2263202_02_Hfq_FLAG_div_by_425675.0_multi_by_1000000.0_reverse.wig.gz |
984.4 Kb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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