CBA × C57BL/6 F1 (CBF1) mice were infected with 400 L3 infective-stage H. bakeri larvae by gavage and adult nematodes were collected from the small intestine 14 days post infection. The nematodes were washed and maintained in serum-free medium in vitro as described previously (17 ). For genomic analyses, DNA was collected from adult worms immediately following harvest from the gut, with extensive washing to remove host material followed by purification using Zymo Research Genomic DNA Clean & Concentrator kit following manufacturer’s instructions (further details in Supplementary Methods). RNA from H. bakeri was collected from adult worms in Qiazol (Qiagen) using mechanical disruption with 5 mm stainless steel beads (Qiagen) on a Tissue Lyser II (Qiagen). Caenorhabditis elegans were harvested and flash frozen as in (18 (link)). Total RNA was treated with RNA 5′ Polyphosphatase (Epicenter) following manufacturer’s instructions, before library preparation. Libraries for small RNA sequencing were prepared using the CleanTag small RNA library prep kit (Trilink) according to manufacturer’s instructions.
Rna 5 polyphosphatase
Manufactured by Illumina
Sourced in United States
RNA 5′ polyphosphatase is a laboratory enzyme used to remove the 5′ polyphosphate groups from RNA molecules. This enzyme is useful for preparing RNA samples for downstream applications, such as RNA sequencing or RNA structure analysis, by modifying the 5′ end of the RNA to a monophosphate form.
Automatically generated - may contain errors
Lab products found in correlation
Hiseq 2500, by Illumina (3 mentions)
Cleantag small rna library prep kit, by TriLink (2 mentions)
Truseq small rna library kit, by Illumina (2 mentions)
Terminator 5 phosphate dependent exonuclease, by Illumina (2 mentions)
18 amplification cycles, by TriLink (2 mentions)
T4 rna ligase, by New England Biolabs (2 mentions)
5 mm stainless steel bead, by Qiagen (1 mentions)
Genomic dna clean concentrator kit, by Zymo Research (1 mentions)
Tissuelyser 2, by Qiagen (1 mentions)
Qiazol, by Qiagen (1 mentions)
Truseq small rna kit, by Illumina (1 mentions)
Truseq small rna library preparation, by Illumina (1 mentions)
Hiseq instrument, by Illumina (1 mentions)
Miseq instrument, by Illumina (1 mentions)
Tripure, by Roche (1 mentions)
Tbe page gel, by Thermo Fisher Scientific (1 mentions)
Spin x 0.22μm cellulose acetate filter columns, by Corning (1 mentions)
Hiseq 2500 sequencer, by Illumina (1 mentions)
Miseq benchtop sequencer, by Illumina (1 mentions)
Rnasin rnase inhibitor, by Promega (1 mentions)
17 protocols using rna 5 polyphosphatase
1
Isolation and Analysis of Helminth Parasites
2
Profiling Nuclear Short RNAs in Yeast
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Five micrograms of total RNA was pretreated with either 20 U of TAP (Epicentre) or 20 U of RNA 5′ polyphosphatase (Epicentre) in a volume of 20 µL for 45 min at 37°C or used directly. After standard extraction, RNA was used for TruSeq small RNA library preparation according to the manufacturer's instructions (Illumina), including 15 cycles of PCR. Libraries were sequenced on a MiSeq instrument (Illumina). A detailed description of processing and analysis of the data thus generated are in the Supplemental Material.
Purification of nuclear short RNAs was performed as described (Chen et al. 2013 (link)). Briefly, nuclei of 2 million young adults grown in liquid culture were isolated as described in Ooi et al. (2010) (link), and RNA was extracted using Tripure (Roche). Short capRNA-seq libraries were cloned from 20 µg of nuclear RNA as follows: After size selection for RNAs of 20–100 nt, we performed RNA polyphosphatase (Epibio) treatment followed by Terminator exonuclease (Epibio) treatment and 3′ adapter ligation. After treatment with heat labile alkaline phosphatase (Epibio), capped RNAs were rendered accessible for cloning by TAP treatment. 5′-adapter cloning and library generation were completed as described in the TruSeq small RNA kit (Illumina), and sequencing was performed on a HiSeq instrument (Illumina, SE50).
Purification of nuclear short RNAs was performed as described (Chen et al. 2013 (link)). Briefly, nuclei of 2 million young adults grown in liquid culture were isolated as described in Ooi et al. (2010) (link), and RNA was extracted using Tripure (Roche). Short capRNA-seq libraries were cloned from 20 µg of nuclear RNA as follows: After size selection for RNAs of 20–100 nt, we performed RNA polyphosphatase (Epibio) treatment followed by Terminator exonuclease (Epibio) treatment and 3′ adapter ligation. After treatment with heat labile alkaline phosphatase (Epibio), capped RNAs were rendered accessible for cloning by TAP treatment. 5′-adapter cloning and library generation were completed as described in the TruSeq small RNA kit (Illumina), and sequencing was performed on a HiSeq instrument (Illumina, SE50).
3
Small RNA Library Preparation
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cDNA libraries were prepared by treating 1–5 μg total RNA or RNA extracted after HRDE-1 immunoprecipitation with 20 Units RNA 5′ polyphosphatase (Epicentre) in a total volume of 20 μl. De-phophorylated RNA was purified by phenol-chloroform extraction and ethanol precipitation according to standard protocols. Subsequent library preparations were performed with the TruSeq Small RNA library kit (Illumina) following the manufacturer’s instructions with exception that 15 cycles of PCR amplification were used. We size-selected cDNA libraries using 6% TBE PAGE gels (Life Technologies) and ethidium bromide staining. Desired sizes of cDNA bands were cut from the gel (between 147 and 157 nt), the gel matrix broken by centrifugation through gel breaker tubes (IST Engineering Inc.), and cDNA eluted with 400 μl of 0.3M Na-Chloride. Further purification of cDNA was by centrifugation through Spin-X 0.22μm cellulose acetate filter columns (Costar) followed by ethanol precipitation. Libraries were sequenced on a MiSeq Benchtop Sequencer or a HiSeq 2500 Sequencer (Illumina).
4
Isolation and Characterization of Trichuris muris Extracellular Vesicles
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Trichuris muris EVs were purified from the ES products of T. muris as described previously (Shears et al., 2018 ), then EVs were isolated by ultracentrifugation (UC), and concentrated using a Vivaspin 6 spin 5 kDa MWCO column. The size and concentration of EVs were assessed by Nanoparticle Tracking Analysis using the NanoSight LM10, and the quality of EVs was assessed by transmission electron microscopy (TEM), which is described in Duque-Correa et al. (2020) . RNA was extracted using the miRNeasy mini kit (Qiagen). For samples that were DNase treated, these were incubated for 10 min at room temperature (RT) with RNase-free DNase I with additional RNasin RNase inhibitor (Promega). Polyphosphatase treatment was performed using RNA 5′ polyphosphatase (Epicentre) following the manufacturer’s instructions. RNA that was enzymatically treated, either with DNase or 5′ polyphosphatase, was subsequently purified by ethanol precipitation. Libraries were prepared using the CleanTag small RNA Library prep kit (TriLink) following the manufacturer’s instructions. Adapters were diluted 1:12 and 18 PCR cycles were used.
5
5'-end RNA Probing via dRNA-Seq
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The 5′-end of the RNA was probed using dRNA-Seq, as previously described (5 (link)). Briefly, rRNA-subtracted RNA was split into two samples. One sample was treated with 20 U RNA 5′ polyphosphatase (Epicentre, Madison, WI, USA) at 37°C for 60 min. The other sample was treated with nuclease-free water. The dephosphorylated RNA adaptor was ligated to both samples using 5 U of T4 RNA ligase (Epicentre) at 37°C for 90 min. The adaptor-ligated RNA samples were purified, and cDNA was synthesized using the SuperScript III First-Strand Synthesis System with 3.125 pmol random nonamers. DNA libraries were amplified by PCR with Phusion High-Fidelity DNA Polymerase, using P5 and P7 index primers for 20 cycles. The amplified libraries were sequenced via the 50 cycles single-ended recipe on a HiSeq 2500 sequencer. The oligonucleotide sequences are summarized in Supplementary Table S1 .
6
RNA-seq Analysis of Small RNAs in Bacteria
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RNA-seq was performed on two independently prepared samples from early-exponential phase cells. Sequencing libraries were prepared from each RNA preparation without fragmentation by using the NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (New England Biolabs, Ipswich, MA, USA) according to the manufacturer’s protocol, with a minor modification. This kit is specialized for sequencing eukaryotic sRNAs that contain a 5′ monophosphate, such as miRNA and siRNA. Therefore, to analyse sRNAs containing a 5′ monophosphate terminus or a triphosphate terminus, the total RNA was treated with RNA 5′ poly phosphatase (Epicentre, Madison, WI, USA) according to the manufacturer’s instructions before a sequencing library was prepared (Fig. 1 ). Paired-end sequencing (2 × 300 bp) was performed on the MiSeq platform (Illumina, San Diego, CA, USA) according to the manufacturer’s protocol. After removal of the low-quality reads and trimming of the adaptor sequences using the FASTX-Toolkit (http://hannonlab.cshl.edu/fastx_toolkit/index.html ), the sequence reads were strand-specifically mapped to the HK1651 genome sequence by using the Burrows–Wheeler Aligner,21 (link) and the data were visualized with Integrative Genomics Viewer.22 (link) The sequence reads obtained have been deposited in the GenBank/EMBL/DDBJ database (DRA submission number, DRA005568; DRR accession number, DRR088289).
7
Profiling C. elegans Small RNAs
8
Single-cell RNA-seq of C. elegans nematodes
9
Affinity Purification of Radiolabeled RNA
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Gel-purified radiolabeled RNA fragments obtained using in vitro transcription were treated with RNA 5′ Polyphosphatase (Epicentre) according to the manufacturer's protocol, purified by phenol:chlorophorm extraction and EtOH precipitation, and resuspended in RNase free water. Radiolabeled RNA (30–40 cps) was incubated with 1.2 µg of Dmr1-His6 fusion protein purified by metal affinity and size exclusion or with 1.2 µg of BSA (negative control) in 14 µL of reaction buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 4 mM DTT, 2 mM MgCl2, 2 mM sodium phosphate pH 7.0) with 1 U/μL of RiboLock RNase inhibitor (Thermo Fisher) for 20 min. on ice. 0.5 U of Terminator 5′-Phosphate-Dependent Exonuclease (Epicentre) was then added, and the reaction incubated for 30 min at 30°C.
10
sRNA Library Preparation and Sequencing
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sRNA libraries were prepared from total RNA as described previously (40 (link)). Briefly, 5 μg of DNase-treated total RNA was subjected to rRNA removal (RiboZero rRNA removal for Gram-positive bacteria; Illumina). rRNA-depleted samples were then subjected to RNA fragmentation using the Ambion RNA fragmentation kit (AM8740). Fragmented RNA was subjected to RNA 5′-polyphosphatase (Epicenter) treatment, which was performed to facilitate the 5′-adapter ligation step. Small RNA libraries were generated by Macrogen using the TruSeq small RNA library kit (Illumina). Then, 100-bp paired-end read sequencing was performed using an Illumina HiSeq2000 sequencer.
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