T7 RNAP was purchased from Beijing Biolink Biotechnology (NEB, Beijing, China). RNase inhibitor was bought from Vazyme Biotech. The NTP mixtures were from Shenggong Co. (Beijing, China). DTT was obtained from Solarbio Life Sciences, and l -T was from Hanwei BioTechnologies (AnhuiWuhu, China). Normal deoxynucleotide phosphoramidites for automated DNA synthesis were obtained from Anhui WuhuHuaren Biotechnique.
Rnase inhibitor
Manufactured by Vazyme
Sourced in China
RNase inhibitor is a laboratory reagent that inactivates ribonuclease (RNase) enzymes. RNase inhibitors help prevent the degradation of RNA samples during experimental procedures.
Automatically generated - may contain errors
Lab products found in correlation
Streptavidin magnetic beads, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Harringtonine, by Solarbio (2 mentions)
Cycloheximide (chx), by Merck Group (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Dithiothreitol (dtt), by Solarbio (1 mentions)
Control probe, by RiboBio (1 mentions)
Mz 16a stereomicroscope, by Leica (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Sybr green, by Thermo Fisher Scientific (1 mentions)
Bst dna polymerase, by New England Biolabs (1 mentions)
9 protocols using rnase inhibitor
1
T7 RNAP-Based Transcription Protocol
2
Biotinylated miRNA Affinity Purification
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Cells were transfected with 80 nM biotinylated miR-23a/b mimics (miR-23a/b probes) or control probe (RiBoBio, Guangzhou, China) and were then harvested in lysis buffer (20 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 0.5% NP40 and 200 U/ml RNase inhibitor (Vazyme)). The lysate was next incubated with streptavidin magnetic beads (Invitrogen) at 4 °C overnight with constant rotation. After incubation, the beads were washed with lysis buffer four times, and RNA was extracted with TRIzol (Invitrogen) for analysis.
3
DNA/RNA Sequence Preparation Protocol
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All the DNA and RNA sequences (listed in Table S1 ) were obtained from Tsingke company (Beijing, China). The oligonucleotides were dissolved in water and heated at 95 ℃ for 5 min and cooled down to room temperature before use. If not specifically mentioned, the oligonucleotides were diluted to the desired concentrations by using the buffer (10 mM Tris–HCl, 50 mM K+, pH 7.9). ThT was purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Duplex-specific nuclease (DSN) was purchased from NEWBORNCO., Ltd. (Shenzhen, China). RNase inhibitor was obtained from Vazyme (Jiangsu, China).
4
Ribosome Profiling Protocol for Translational Efficiency Analysis
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Ribo‐seq was performed as previously described [33 (link)]. SCC15 cells were first incubated with harringtonine (Solarbio) at a concentration of 2 μg/mL for 2 min, and then 100 μg/mL cycloheximide (Sigma‐Aldrich) was used to block translation. To prepare ribosome footprints, 300 μL lysate was mixed with 7.5 μL RNase I and 5 μL DNase I and incubated at 25°C for 45 min later. Nuclease digestion was stopped by adding 10 μL RNase inhibitor (Vazyme). Then, 100 μL of digested ribosome footprints was added to the column and centrifuged at 600 ×g for 2 min. Ribo‐seq libraries were constructed using NEBNext® Multiple Small RNA Library Prep Set for Illumina® (New England Biolabs).
The Ribo‐seq data were analyzed using the RiboTool kit (https://bioinformatics.sc.cn/RiboToolkit ) [34 (link)]. The translation efficiency (TE) was calculated as the ratio between the numbers of ribosome fragments aligned to the sequence coding for amino acids in protein (CDS) region and mRNA abundance for individual genes. Codon frequency analysis was performed after eliminating the genes with mRNA abundances lower than 5, and TEs with a 1‐fold decrease were considered TE‐downregulated genes.
The Ribo‐seq data were analyzed using the RiboTool kit (
5
Wasp Venom Apparatus RNA Extraction
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Mated female wasps aged 2–7 days were chilled at 4 °C for 10 min and then rinsed in sterile phosphate-buffered saline (PBS, pH 7.2) followed by dissection in PBS with 1 unit/μL RNase inhibitor (Vazyme, Nanjing, China) on an ice plate under a Leica MZ 16A stereomicroscope (Leica, Wetzlar, Germany). The venom apparatus and carcass (minus the venom apparatus) were collected in 1 mL of TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Total RNA was extracted as per the manufacturer’s protocol. The quantity of the total RNA samples was determined by NanoDrop 2000 (Thermo Scientific, Wilmington, DE, USA) and stored at −80 °C for subsequent experiments.
6
Oasl2-209 lncRNA Pulldown Protocol
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The 3'-terminal biotinylated probe complementary to the long noncoding RNA (lncRNA) Oasl2-209 sequence was synthesized, and a scrambled biotinylated probe was used as the negative control (Genescript, Nanjing, China). These probes (10 pmol/µl) were then incubated with streptavidin magnetic beads (Invitrogen) for 2 h with constant rotation. Cells were harvested in lysis buffer (20 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 0.5% NP40, and 200 U/ml RNase inhibitor (Vazyme)). Then, the lysates were incubated with the probe-coated beads at 4 °C overnight with constant rotation. After incubation, the beads were washed with wash buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, and 2 M NaCl) three times, and RNA was extracted with TRIzol (Invitrogen) for analysis. The sequences of the probes were as follows: Oasl2-209 probe, 5'-CCC AAA TTA TCA TTA CCA CC-3'; control probe, 5'-TGA TGT CTA GCG CTT GGG CTT TG-3'.
7
Quantifying rRNA Methylation Levels
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The levels of rRNA methylation were analyzed by RT-QPCR68 (link). A reverse-transcription primer R and a pair of primers (F1 and R1) near A674 on 26S rRNA were designed for QPCR. The sequences of the primers were R: 5′-AGTCACAAGTGACACGCAC-3′; F1: 5′-ACAGTGTTGCCCATCTCGC-3′; R1: 5′-ACGTCGGCCAATTCGAGAC-3′. The 26S rRNA transcript was specifically reversed transcribed using primer R to generate cDNA with 100 ng of total RNA extracted from either WT or rrp-8(kun54) mutant worms and either 10 μM or 1 mM dNTPs in each sample. The reverse transcription system included 200U Hscript Reverse Transcriptase (Vazyme Biotech), 40U RNase inhibitor (Vazyme Biotech), 1 μM specific reverse primer R, and either 10 μM and 1 mM dNTP in each sample. For each reaction, reverse transcription was performed at 25 ℃ for 5 min, followed by an incubation at 50 ℃ for 15 min and at 85 ℃ for 5 min. RT-QPCR was performed to quantify the specific cDNA levels using the primer pair F1/R1 spanning the methylation site. The relative methylation rate of a specific site on 26S rRNA was calculated as the ratio of the cDNA levels obtained using 1 mM dNTPs to that obtained using 10 μM dNTPs in each sample.
8
Ribosome Sequencing for Translational Profiling
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Ribosome sequencing (Ribo-Seq) was performed as previously described (22 (link)). Briefly, SCC25 cells were incubated with harringtonine (Solarbio), and then cycloheximide (Sigma-Aldrich) was used to block translation. After incubation with RNase I and DNase I at 25°C for 45 minutes, nuclease digestion was stopped with RNase inhibitor (Vazyme). Then, digested ribosome footprints were added to the column and centrifuged at 600g for 2 minutes. Ribo-Seq libraries were constructed using NEBNext R Multiple Small RNA Library Prep Set for Illumina R (New England Biolabs). The Ribo-Seq data were analyzed using the RiboTool kit (https://bioinformatics.sc.cn/RiboToolkit ). Translation efficiency (TE) ratios between the numbers of ribosome fragments were calculated consistent with the sequence coding for amino acids in protein (CDS) region and mRNA abundance for individual genes. TEs with a 1-fold decrease were considered TE-downregulated genes.
9
Multiplex RT-LAMP Virus Detection
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First of all, the primers corresponding to different viruses were pre-immobilized in eight reaction chambers by heating drying. Six micro chambers were employed for detections of three viruses with one repetition, and the remaining two were used for negative controls (Fig. 1 B). The related primer mixture was pre-loaded into the reaction holes. Secondly, the real-time RT-LAMP reaction solution was prepared in a volume of 10.2 μL comprising 50 μM SYBR Green (Invitrogen,USA), 800 U/μL of Bst DNA polymerase (New England BioLabs, USA), 5000 U/mL of AMV transcripitase (New England BioLabs, USA) and 40 U/μL RNase inhibitor (Vazyme, China). The premixed reaction solution containing 14.8 μL of nucleic acid was injected into sample wells of the microfluidic chip directly by pipette, and then sample wells and vent wells were sealed by the film. The preprocessed microfluidic chip was then placed in the provided microfluidic device to react at 63.5 °C for 60 min. To ensure the accuracy of the three LAMP systems, the amplification products were further determined by standard agarose gel electrophoresis.
Generally, the amplification result was regarded as positive only when experiments with one repetition shows a positive signal and the negative controls show negative signals in 60 min. A complete set of procedures was showed inFig. 1 C.
Generally, the amplification result was regarded as positive only when experiments with one repetition shows a positive signal and the negative controls show negative signals in 60 min. A complete set of procedures was showed in
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