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| Status |
Public on May 01, 2020 |
| Title |
Hfq CLASH replicate 1 Negative Control Sample |
| Sample type |
SRA |
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| Source name |
Escherichia coli grown in LB
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| Organism |
Escherichia coli str. K-12 substr. MG1655 |
| Characteristics |
5' in-read barcode: NNNCGTGAT
strain: MG1655
optical density: n/a
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| Treatment protocol |
Once cells reached the desired density they were either cross-linked in the Vari-X-linker (22 seconds; ~500mJ/cm2) or harvested by rapid filtration.
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| Growth protocol |
Cells were grown in standard LB medium to the desired optical density (OD600).
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using the Guanidium thiocyanate phenol method. RNA integrity was assessed with the Prokaryote Total RNA Nano assay on a 2100 Bioanalyzer (Agilent). Genomic DNA was removed by incubating 10 μg of total RNA with 2U Turbo DNase (Ambion) in a 50 μl final volume for 30 minutes at 37°C in the presence of 10 U SuperaseIn RNase Inhibitor (Ambion). RNA was subsequently phenol-chloroform extracted and purified by ethanol-precipitation.
For CLASH: CLASH was performed essentially as described (Waters et al., 2017), with a number of modifications including changes in incubation steps, cDNA library preparation, reaction volumes and UV cross-linking. E. coli expressing the chromosomal Hfq-HTF were grown overnight in LB at 37°C with shaking (200 rpm), diluted to starter OD600 0.05 in fresh LB, and re-grown with shaking at 37°C in 750 ml LB. A volume of culture equivalent to 80 OD600 per ml was removed at the following cell-densities (OD600): 0.4, 0.8, 1.2, 1.8, 2.4, 3.0 and 4.0, and immediately subjected to UV (254 nm) irradiation for 22 seconds (~500 mJ/cm2) in the Vari-X-linker (Van Nues et al., 2017a). Cells were harvested using a rapid filtration device (Van Nues et al., 2017a) onto 0.45 μM nitrocellulose filters (Merck Millipore) and flash-frozen on the membrane in liquid nitrogen. The following day, the membranes were washed with ~15 ml ice-cold phosphate-buffered saline (PBS), and cells were harvested by centrifugation. Cell pellets were lysed by bead-beating in 1 volume per weight TN150 buffer (50mM Tris pH 8.0, 150 mM NaCl, 0.1% NP-40, 5 mM β-mercaptoethanol) in the presence of protease inhibitors (Roche), and 3 volumes 0.1 mm Zirconia beads (Thistle Scientific), by performing 5 cycles of 1 minute vortexing followed by 1-minute incubation on ice. One additional volume of TN150 buffer was added. To reduce the viscosity of the lysate and remove contaminating DNA the lysate was incubated with RQ1 DNase I (10U/ml Promega) for 30 minutes on ice. Two-additional volumes of TN150 were added and mixed with the lysates by vortexing. The lysates were centrifuged for 20 minutes at 4000 rpm at 4°C and subsequently clarified by a second centrifugation step at 13.4 krpm, for 20 min at 4°C. Purification of the UV cross-linked Hfq-HTF-RNA complexes and cDNA library preparation was performed as described (Granneman et al., 2009b). Cell lysates were incubated with 50 μl of pre-equilibrated M2 anti-FLAG beads (Sigma) for 1-2 hours at 4°C. The anti-FLAG beads were washed three times 10 minutes with 2 ml TN1000 (50 mM Tris pH 7.5, 0.1% NP-40, 1M NaCl) and three times 10 minutes with TN150 without protease inhibitors (50 mM Tris pH 7.5, 0.1% NP-40, 150mM NaCl). For TEV cleavage, the beads were resuspended in 250 μl of TN150 buffer (without protease inhibitors) and incubated with home-made GST-TEV protease at room temperature for 1.5 hours. The TEV eluates were then incubated with a fresh 1:100 dilution preparation of RNaceIt (RNase A and T1 mixture; Agilent) for exactly 5 minutes at 37ºC, after which they were mixed with 0.4g GuHCl (Sigma), NaCl and Imidazole to final concentrations of 6M, 300 mM and 10 mM, respectively. Note this needs to be carefully optimized to obtain high-quality cDNA libraries. The samples were then transferred to 50 μl Nickel-NTA agarose beads (Qiagen), equilibrated with wash buffer 1 (6 M GuHcl, 0.1% NP-40, 300 mM NaCl, 50 mM Tris pH 7.8, 10 mM Imidazole, 5 mM beta-mercaptoethanol). Binding was performed at 4ºC overnight with rotation. The following day, the beads were transferred to Pierce SnapCap spin columns (Thermo Fisher), washed 3 times with wash buffer 1 and 3 times with 1xPNK buffer (10 mM MgCl2, 50mM Tris pH 7.8, 0.1% NP-40, 5 mM beta-mercaptoethanol). The washes were followed by on-column TSAP incubation (Thermosensitive alkaline phosphatase, Promega) treatment for 1h at 37ºC with 8 U of phosphatase in 60 μl of 1xPNK, in the presence of 80U RNasin (Promega). The beads were washed once with 500 μl wash buffer 1 and three times with 500 μl 1xPNK buffer. To add 3’-linkers (App-PE - Table S7), the Nickel-NTA beads were incubated in 80 μl 3’-linker ligation mix with (1 X PNK buffer, 1 mM 3’-adapter, 10% PEG8000, 30U Truncated T4 RNA ligase 2 K227Q (NEB), 60U RNasin). The samples were incubated for 4 hours at 25ºC. The 5’-ends of bound RNAs were radiolabeled with 30U T4 PNK (NEB) and 3μl 32P-γATP (Perkin Elmer) in 1xPNK buffer for 40 min at 37ºC, after which 100 mM cold ATP (Roche) was added to a final concentration of 1mM, and the incubation prolonged for another 20 min to complete 5’-end phosphorylation. The resin was washed three times with 500 μl wash buffer 1 and three times with equal volume of 1xPNK buffer. For on-bead 5’-linker ligation, the beads were incubated 16h at 16ºC in 1xPNK buffer with 40U T4 RNA ligase I (NEB), and 1 μl 100 μM L5 adapter (Table S7), in the presence of 1mM ATP and 60U RNasin (Promega). The Nickel-NTA beads were washed three times with wash buffer 1 and three times with buffer 2 (50 mM Tris–HCl pH 7.8, 50 mM NaCl, 10 mM imidazole, 0.1% NP-40, 5 mM β-mercaptoethanol). The protein-RNA complexes were eluted in two steps in new tubes with 200 μl of elution buffer (wash buffer 2 with 250 mM imidazole). The protein-RNA complexes were precipitated on ice by adding TCA to a final concentration of 20%, followed by a 20-minute centrifugation at 4ºC at 13.4 krpm. Pellets were washed with 800 μl acetone, and air dried for a few minutes in the hood. The protein pellet was resuspended and incubated at 65ºC in 20 μl 1x NuPage loading buffer (Novex), resolved on 4–12% NuPAGE gels, and visualised by autoradiography. The cross-linked proteins-RNA were cut directly from the gel and incubated with 160 μg of Proteinase K (Roche) in 600 μl wash buffer 2 supplemented with 1% SDS and 5 mM EDTA at 55ºC for 2-3 hours with mixing. The RNA was subsequently extracted by phenol-chloroform extraction and ethanol precipitated. The RNA pellet was directly resuspended in RT buffer and was transcribed in a single reaction with the SuperScript IV system (Invitrogen) according to manufacturer’s instructions using the PE_reverse oligo as primer. The cDNA was purified with the DNA Clean and Concentrator 5 kit (Zymo Research) and eluted in 11 μl DEPC water. Half of the cDNA (5 μl) was amplified by PCR using Pfu Polymerase (Promega) with the cycling conditions (95°C for 2 min; 20-24 cycles: 95°C for 20s, 52°C for 30s and 72°C for 1 min; final extension of 72°C for 5 min). The PCR primers are listed in Table S7. PCR products were treated with 40U Exonuclease 1 (NEB) for 1 h at 37ºC to remove free oligonucleotide and purified by ethanol precipitation/ or the DNA Clean and Concentrator 5 kit (Zymo Research). Libraries were resolved on a 2% MetaPhor agarose (Lonza) gel and 175-300bp fragments were gel-extracted with the MinElute kit (Qiagen) according to manufacturer’s instructions. All libraries were quantified on a 2100 Bionalyzer using the High-Sensitivity DNA assay. Individual libraries were pooled based on concentration and barcode sequence identity. Paired-end sequencing (75 bp) was performed by Edinburgh Genomics on an Illumina HiSeq 4000 platform.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
CLASH sample
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| Data processing |
Library strategy: CRAC (CLIP-Seq derivative)
Raw sequencing reads in fastq files were processed using a pipeline developed by Sander Granneman, which uses tools from the pyCRAC package (Webb et al., 2014a). The entire pipeline is available at https://bitbucket.org/sgrann/). The CRAC_pipeline_PE.py pipeline first demultiplexes the data using pyBarcodeFilter.py and the in-read barcode sequences found in the L5 5’ adapters. Flexbar then trims the reads to remove 3’-adapter sequences and poor quality nucleotides (Phred score <23). Using the random nucleotide information present in the L5 5’-adaptor sequences, the reads were collapsed to remove potential PCR duplicates. The reads were then mapped to the Escherichia coli MG1655 genome with Novoalign (www.novocraft.com). To determine which genes the reads overlapped with we generated an annotation file in the Gene Transfer Format (GTF). This file contains the start and end positions of each gene on the chromosome as well as what genomic features (i.e. sRNA, protein- coding, tRNA) it belongs to. To generate this file, we used the Rockhopper software (Tjaden, 2015) on E. coli rRNA-depleted total RNA-seq data (generated by Christel Sirocchi), a minimal GTF file obtained from ENSEMBL (without UTR information). The resulting GTF file contained information not only on the coding sequences, but also complete 5’ and 3’ UTR coordinates. PyReadCounters then used the novoalign output file and the GTF file to count the total number of unique cDNAs that mapped to each gene.
To demultiplex the CLASH datasets, the following command lines were used: pyBarcodeFilter.py -f Hfq_CRAC_1_1.fastq -r Hfq_CRAC_1_2.fastq -k -m 1 -b barcodes_CLASH1.txt & pyBarcodeFilter.py -f Hfq_CRAC_2_1.fastq -r Hfq_CRAC_2_2.fastq -k -m 1 -b barcodes_CLASH2.txt &. NOTE!! The -k flag in these command lines instructs pyBarcodeFilter NOT to remove the 5' in-read barcodes. Therefore, before analyzing the data, the user still needs to trim these sequences from the Forward read CLASH fastq files.
Genome_build: Escherichia_coli_str_k_12_substr_mg1655.GCA_000005845.2.22
Supplementary_files_format_and_content: text files with raw counts or TPM. GTF and FASTA files are available on the SuperSeries record.
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| Submission date |
Nov 28, 2018 |
| Last update date |
May 01, 2020 |
| Contact name |
Sander Granneman |
| E-mail(s) |
Sander.Granneman@ed.ac.uk
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| Organization name |
University of Edinburgh
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| Department |
Centre for Synthetic and Systems Biology
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| Lab |
Granneman lab
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| Street address |
Mayfield Road, Kings Buildings, Waddington building, room 3.06
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| City |
Edinburgh |
| ZIP/Postal code |
EH9 3JD |
| Country |
United Kingdom |
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| Platform ID |
GPL24659 |
| Series (2) |
| GSE123049 |
Hfq CLASH uncovers sRNA-target interaction networks linked to nutrient availability adaptation [CLASH] |
| GSE123050 |
Hfq CLASH uncovers sRNA-target interaction networks linked to nutrient availability adaptation |
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| Relations |
| BioSample |
SAMN10492628 |
| SRA |
SRX5078332 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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