Purified RNAs were heated at 75 °C for 3 min to avoid biases likely caused by the aggregations and secondary structures of RNAs11 (link),31 (link). Yeast poly(A) polymerase (Thermo Fisher) was used to add the poly(A) tail to RNA. Specifically, 0.1 µg/µl RNA was incubated at 37 °C for 1 h in a 20 mM Tris-HCl (pH 7.0) buffer with 20 μM EDTA, 0.2 mM DTT, 0.6 mM MnCl2, 10% glycerol, 100 μg/ml acetylated BSA, 2.5 mM rATP, 40 U/µl Yeast poly(A) polymerase (Thermo Fisher), and 0.5 U/µl RNase inhibitor (SUPERase.InTm, Thermo Fisher). The reaction was terminated by heating at 65 °C for 15 min. It was reported that the 3′-terminal nucleotide influences the poly(A) tail efficiency11 (link). In our method, the capturing efficiency is not expected to vary significantly once the poly(A) tail is longer than 30 nt (the length of the complementary DNA use for capturing miRNAs). Furthermore, the influences of the 3′-terminal nucleotide on miRNA capturing efficiency could be effectively avoided by maintaining the poly(A) tailing reaction for 1 h (ref. 32 (link)).
Yeast poly a polymerase
Manufactured by Thermo Fisher Scientific
Yeast poly(A) polymerase is an enzyme that catalyzes the addition of a polyadenosine (poly(A)) tail to the 3' end of messenger RNA (mRNA) molecules. This process is essential for the stability and translation of mRNA in eukaryotic cells.
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Lab products found in correlation
Qubit fluorometer, by Thermo Fisher Scientific (2 mentions)
2nd generation 5 3 race kit, by Roche (1 mentions)
Qiaquick nucleotide removal kit, by Qiagen (1 mentions)
Tdt buffer, by Thermo Fisher Scientific (1 mentions)
Superscript 3, by Thermo Fisher Scientific (1 mentions)
Smarter race 5 3 kit, by Takara Bio (1 mentions)
Rnase t1, by Merck Group (1 mentions)
Rnase a, by Merck Group (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Pseudouridine triphosphate, by TriLink (1 mentions)
T7 mscript standard mrna production system, by CellScript (1 mentions)
5 methylcytidine triphosphate, by TriLink (1 mentions)
Megaclear transcription clean up kit, by Thermo Fisher Scientific (1 mentions)
3 0 me m7g 5 ppp 5 g arca cap analog, by TriLink (1 mentions)
Antarctic phosphatase, by New England Biolabs (1 mentions)
Turbo dnase, by Thermo Fisher Scientific (1 mentions)
α 32p cordycepin 5 triphosphate, by PerkinElmer (1 mentions)
Typhoon fla 7000, by GE Healthcare (1 mentions)
Multi gauge software, by Fujifilm (1 mentions)
Igor pro, by Wavemetrics (1 mentions)
11 protocols using yeast poly a polymerase
1
Poly(A) Tailing of RNA for miRNA Capture
2
Comprehensive ZIKV Genome Sequencing
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ZIKV cDNA amplicons were subjected to Sanger sequencing with overlapping reads that covered the amplicons on both strands (primer sequences available upon request). For sequencing of the 5′ and 3′ termini of the genome, isolated ZIKV RNA was first polyadenylated using yeast poly(A) polymerase (Affymetrix), followed by rapid amplification of cDNA ends (RACE). RACE was done with the 2nd Generation 5′/3′ RACE kit (Roche) using primers SP1, SP2, SP3, SP5 (Table S1 ) and the primers included in the kit, according to the manufacturer’s instructions. The 5′ and 3′ amplicons were sequenced using primers 5RACE-seq1 and 3RACE-seq, respectively (Table S1 ).
For NGS, the 5 amplicons were gel-purified, pooled in equimolar concentrations and library preparation was performed according to the Pacific Biosciences Amplicon Template Preparation protocol. Sequencing was performed on a single SMRT cell using a 6-hour runtime and the C4-P6 chemistry on the RSII platform. Reads-of-inserts with a minimum of 5 full passes and minimum predicted accuracy of 99% were subsequently mapped to the ZIKV SL1602 reference sequence using SMRT Analysis software version 2.3. For each amplicon, primer sequences were removed and nucleotide counts were generated for each genomic position.
For NGS, the 5 amplicons were gel-purified, pooled in equimolar concentrations and library preparation was performed according to the Pacific Biosciences Amplicon Template Preparation protocol. Sequencing was performed on a single SMRT cell using a 6-hour runtime and the C4-P6 chemistry on the RSII platform. Reads-of-inserts with a minimum of 5 full passes and minimum predicted accuracy of 99% were subsequently mapped to the ZIKV SL1602 reference sequence using SMRT Analysis software version 2.3. For each amplicon, primer sequences were removed and nucleotide counts were generated for each genomic position.
3
Fluorescent Labeling of RNA Precursor
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To label the 3΄-end of precursor 1 using Terminal Deoxynucleotidyl Transferase (TdT), DNA has to be attached to its 3΄-end because 3΄ terminal ribonucleotides are poorly accepted by the enzyme. Addition of a poly-deoxyadenosine (poly-dA) tail to the 3΄-end of precursor 1 was done in a final volume of 25 μl containing 60 pmol of precursor 1, 1× Poly(A) Polymerase reaction buffer (Affymetrix), 0.5 mM deoxyadenosine triphosphate (dATP) (Life Technologies) and 600 units of Yeast Poly(A) Polymerase (Affymetrix). The reaction was incubated at 37°C for 8 h and was column-purified using the QIAquick Nucleotide Removal Kit according to the manufacturer's instructions (Qiagen). The eluted oligonucleotide was ethanol precipitated and desalted by drop dialysis. The 3΄-end labeling of precursor 1 was done in a final volume of 30 μl containing 1× TdT buffer (Life Technologies), 100 μM fluorescein-12-uridine triphosphate (Fluorescein-12-UTP) (Roche Life Science) and 30 units of TdT (Life Technologies). The reaction was incubated at 37°C for 60 min and the 3΄-fluorescein-labeled precursor 1 was phenol/chloroform extracted, ethanol precipitated and gel-purified.
4
Determination of BVDV Genome 5' End
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Determination of the 5′ end of the BVDV genome was done using SMARTer RACE 5′/3′ Kit (Clontech, Takarabio). Briefly, RNA was purified from 250 μl viral supernatant as described above and cDNA was produced according to manufacturer’s instructions using a BVDV specific primer (TS-O-00039). The cDNA was amplified using 10×UPM and TS-O-01059 with a touchdown PCR with cycling parameters of 5 cycles of 94°C for 30 s, 64°C for 30 s and 72°C for 2 min, followed by 5 cycles of 94°C for 30 s, 62°C for 30 s and 72°C for 2 min, followed by 30 cycles of 94°C for 30 s, 60°C for 30 s and 72°C for 2 min. For 3′ end determination, BVDV RNA was tailed with ATP (10 mM) using Yeast Poly(A) Polymerase (Affymetrix, Thermo Fisher Scientific) at 37°C for 10 min and cDNA was made using SuperScript III (Life Technologies) and a custom-made primer with a poly-T tail (TS-O-00178) in a gradient from 48 up to 55°C, during 1 h. The cDNA was amplified using AUAP and TS-O-00913 and the product was purified with Zymoclean Gel DNA Recovery. Primers for the described procedures are listed in Supplementary Table S2 .
5
Quantifying Uncleaved and Cleaved MALAT1
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To specifically detect the levels of uncleaved and cleaved MALAT1, RNA samples were subject to PolyG tailing, before being reverse transcribed with Oligo dC primers tagged with an adaptor sequence. Poly G tailing was performed using yeast poly(A) polymerase (Affymetrix) and rGTP. After reverse transcription, qPCR was performed using the adaptor as common reverse primer, and gene-specific forward primers located upstream and downstream of mascRNA site to detect cleaved and uncleaved MALAT1, respectively. For internal control of RNA loading and reaction efficiency, a forward primer located at the 3′ end of GAPDH mRNA was used. Sequences of primers are listed in supplementary methods.
6
Poly(A) Tail Size Distribution Analysis
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Total RNA was isolated from the cells using the RNeasy Mini kit (QIAGEN). Poly(A) tail size distribution profile analysis (Lingner and Keller 1993 (link)) was performed as described previously (Huang et al. 2013 (link)). Briefly, total RNA was end-labeled with [α-32P]3′-cordycepin using yeast poly(A) polymerase (Affymetrix). Labeled RNA was then digested with RNase T1 (Sigma-Aldrich) and RNase A (Sigma-Aldrich). Radioactivity was measured after precipitation with trichloroacetic acid, and 20,000 CPM of each sample was used for electrophoresis on 8% denaturing polyacrylamide–urea gels. Autoradiograms were scanned and analyzed using ImageJ software (NIH) to obtain poly(A) length-distribution profiles. All the poly(A) tail distribution profiling assays were repeated at least twice.
7
Synthetic mRNA-Cre Construct Production
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The syn-mRNA-Cre construct (Fig 1C ) was synthesized in vitro using the T7 mScript Standard mRNA Production System (CELLSCRIPT, Madison, WI) and 2 μg of purified DNA template. The template DNA was designed (S4 Fig ) and synthesized using AAV-pgk-Cre, a kind gift from Patrick Aebischer (Addgene plasmid # 24593). A custom ribonucleotide blend comprised of 3′-0-Me-m7G(5′)ppp(5′)G ARCA cap analog, pseudouridine triphosphate, 5-methylcytidine triphosphate (TriLink Biotechnologies, San Diego, CA), ATP, and GTP (New England Biolabs) was prepared. The final reaction mixture (20 μL), containing 6 mM ARCA cap analog, 3.0 mM ATP, and 1.5 mM of each of the other nucleotides, was incubated for 1 hour at 37°C. The DNA template was then degraded by Turbo DNase (Life Technologies, Grand Island, NY), which was removed by ammonium acetate precipitation. The residual 5′-triphosphates were degraded by 2 hour incubation at 37°C with Antarctic phosphatase (New England Biolabs), which was removed by ammonium acetate precipitation. After a 2 hour treatment at 37°C with yeast Poly(A) Polymerase (Affymetrix, Santa Clara, CA), the polyadenylated synthetic mRNA was finally repurified with a MEGAclear Transcription Clean-Up Kit, diluted with RNAsecure Resuspension Solution and quantified with a Qubit fluorometer (all from Thermo Fisher Scientific).
8
Passenger Strand Cleavage Detection Protocol
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Detection of passenger strand cleavage was performed as described (Matranga et al. 2005 (link)) with the following modifications: The 3′ end of the passenger strand was radiolabeled by [α-32P] cordycepin-5′-triphosphate (PerkinElmer) and yeast poly(A) polymerase (Thermo Fisher Scientific), while the 5′ end of the guide strand was nonradioactively monophosphorylated. Typically, in 20 µL reactions, 2 µL of siRNA duplexes were incubated at 25°C with 10 µL of lysate from HEK293T cells overexpressing FLAG-tagged Ago2, 2 µL of 1 µM 2′-O-methyl oligonucleotide complementary to the passenger strand, and 6 µL of 40× reaction mix. An amount of 2 µL of the reaction mixture was taken at each time point, mixed with 8 µL of low-salt PK solution, and incubated at 55°C for 10 min. An equal volume of 2× formamide dye was then added and incubated at 68°C for 5 min. The 3′ cleavage fragments of the passenger strand were analyzed on an 15% denaturing polyacrylamide gel. Gels were dried and imaged by Typhoon FLA 7000 (GE Healthcare Life Sciences) and quantified using MultiGauge software (Fujifilm Life Sciences). Graphs were prepared using IgorPro (WaveMetrics).
9
3'-RACE for lncTAM34a Identification
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3′-RACE was performed as described as previously in Ref. 8 (link). Briefly, U2OS cell RNA was polyA-tailed using yeast polyA polymerase (ThermoFisher Scientific, Cat# 74225Z25KU) after which cDNA was synthesized using oligo(dT) primers. Nested-PCR was performed first using a forward primer in lncTAM34a exon 1 and a tailed oligo(dT) primer followed by a second PCR using an alternate lncTAM34a exon 1 primer and a reverse primer binding to the tail of the previously used oligo(dT) primer. PCR products were gel purified and cloned the Strata Clone Kit (Agilent Technologies, Santa Clara, CA, USA, Cat# 240205), and sequenced.
10
Poly(G) Tailing for Poly(A) Tail Analysis
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PAT assay was performed by using a poly(G) tailing method as previously described with minor modifications43 (link). Briefly, total RNA was isolated from 500 GV oocytes. Total RNA was incubated with 75 U of yeast poly(A) polymerase (Thermo) in the presence of 0.375 mM guanosine-5′-triphosphate and 0.125 mM inosine triphosphate (at 37 °C for 60 min. cDNA was synthesized by using SuperScript II Reverse Transcriptase (Invitrogen) and PAT-C10T2 primer at 42 °C for 1 h. PAT-PCR was performed using a gene-specific forward primer (Supplementary Table 2 ) and PAT-PCR-R prime or gene-specific reverse (A0) primer with KAPA HiFi HotStart ReadyMix (KAPA Biosystems). Due to additional G tailing and adaptor sequence, the length of polyadenylation PCR products minus A0 products is at least 35 bp longer than the actual poly(A) tails. PCR products were resolved in a 2% agarose gel. The size of PCR products was also analyzed with Fragment Analyzer (Advanced Analytical).
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