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| Status |
Public on Jun 01, 2017 |
| Title |
E. coli 3' RACE Rep1 |
| Sample type |
SRA |
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| Source name |
Escherichia coli
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| Organism |
Escherichia coli |
| Characteristics |
strain: K12 MG1655
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using the hot phenol method as described in (Lybecker et al., 2014).
total DNaseI treated RNA was depleted of ribosomal RNA using the Ribo-Zero™ RNA removal kit for Gram-negative bacteria (Epicentre). A 3' RNA adapter, based on the Illumina multiplexing adapter sequence (Oligonucleotide sequences © 2007-2014 Illumina, Inc. All rights reserved) blocked at the 3' end with an inverted dT (5'-GAUCGGAAGAGCACACGUCU[idT]-3'), was phosphorylated at the 5' end using T4 PNK (New England Biolabs) per the manufacturer’s protocol. The 3' RNA adapter was ligated to the 3' ends of the rRNA depleted RNA using T4 RNA ligase I (New England Biolabs). 1.5 mg of RNA was incubated at 20°C for 6 hours in 1X T4 RNA ligase reaction buffer with 1 mM ATP, 30 µM 3' RNA adapter, 10 % DMSO, 10 U of T4 RNA ligase I, and 40 U of RNasin (Promega) in a 20 ml reaction. RNA was then fragmented in equivalents of 100 ng using the RNA fragmentation reagents (Ambion®) per the manufacturer’s protocol at 70°C for 3 min and subsequently phosphorylated at the 5' ends using T4 PNK (New England Biolabs) per the manufacturer’s protocol to allow for ligation of the 5' adapter. RNA was size-selected (≈ 150-300 nt) and purified over a denaturing 8 % polyacrylamide/8 M urea/TBE gel. Gel slices were incubated in RNA elution buffer (10 mM Tris-HCl, pH 7.5, 2 mM EDTA, 0.1 % SDS, 0.3 M NaOAc) with vigorous shaking at 4°C overnight. The supernatant was subsequently ethanol precipitated using glycogen as a carrier molecule. The Illumina small RNA 5' adapter (5'-GUUCAGAGUUCUACAGUCCGACGAUC-3') was ligated to the RNA as described before except the concentration of the adapter was 52 mM and 20 U of T4 RNA ligase I was used in total volume of 25 µl. The ligated RNAs were size-selected (≈ 200-300 nt) and gel-purified over a denaturing 8 % polyacrylamide/8 M urea/TBE gel (as described above). The di-tagged RNA libraries were reverse-transcribed with SuperScript®II reverse transcriptase (Invitrogen) using random nonamers per the manufacturer’s protocol. RNA was removed using RNase H (Promega) per the manufacturer’s protocol and cDNA was amplified in PCR carried out using Phusion® High-Fidelity Polymerase (New England Biolabs). cDNA was amplified with modified designed Illumina-compatible PCR primers (3’ library Forward 5’-CAAGCAGAAGACGGCATACGACAGGTTCAGAGTTCTACAGTCCGA-3’; Reverse 5’-AATGATACGGCGACCACCGAGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC-3’) by 18 cycles of PCR. The products were purified using Agencourt AMPure XP beads (Beckman) and analyzed on an Agilent 2100 Bioanalyzer. 3’ end enriched cDNA libraries were sequenced on individual Genome Analyzer IIx lanes (36 bp, single-end) or on HiSeq 2000 lanes (50 bp, single-end) using primer based on Illumina Multiplexing Read 2 Sequencing Primer (5’-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC-3’) at the CSF NGS unit http://csf.ac.at/.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
RACE |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
RNA (RACE)
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| Data processing |
50 bp single-end reads, were mapped to the genome with using bowtie2(Langmead and Salzberg, 2012) using default parameters. Alignments with bowtie2 mapping quality values lower than 40 were not retained for further analysis, leaving 128,413,654 and 76,508,335 reads.
In order to distinguish between 3’ ends of transient products of RNA metabolism and stable 3’ ends, we developed an algorithm to call coverage peaks. The algorithm will be discussed in detail in an upcoming manuscript, and the scripts used are available upon request from the authors. In short, positions in the E. coli genome were considered in descending order of coverage and assigned a p-value based on a Poisson distribution parameterized by the mean coverage of all covered bases in the genome. Peaks were rejected if their p-values exceeded 1e-4 or if there existed a >10-bp window containing the peak in which all positions were within 2-fold coverage of the peak position. Parameters were chosen based on analysis of annotated 3’ ends as well as qualitative analysis of peaks. This resulted in 20,019 peaks.
Genome_build: NC_000913.3
Supplementary_files_format_and_content: GFF file containing 3' end annotations
Supplementary_files_format_and_content: BIGWIG file containing coverage tracks from 3' RACE data
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| Submission date |
May 08, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Philipp Rescheneder |
| E-mail(s) |
philipp.rescheneder@univie.ac.at
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| Organization name |
MFPL
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| Street address |
Dr. Bohr Gasse 9
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| City |
Vienna |
| ZIP/Postal code |
1030 |
| Country |
Austria |
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| Platform ID |
GPL14548 |
| Series (2) |
| GSE98660 |
Natural RNA polymerase aptamers regulate transcription in E. coli [3' RACE] |
| GSE98661 |
Natural RNA polymerase aptamers regulate transcription in E. coli |
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| Relations |
| BioSample |
SAMN06915666 |
| SRA |
SRX2789658 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2608039_EcoDatK12-3p-ends-r1-minus.bw |
3.3 Mb |
(ftp)(http) |
BW |
| GSM2608039_EcoDatK12-3p-ends-r1-plus.bw |
3.1 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
| Processed data included within Sample table |
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