Cells were grown on 6 well plates or 6 cm dishes. Total RNA used for the analysis of unfractionated mRNAs was extracted from the cells using the RNeasy Plus Mini Kit (QIAGEN, 74136) according to the manufacturer's instructions, except for technical replicate 2 of the samples from RCC4 cells (see Supplementary Table 1). For these samples, cells were lysed with 350 µL of Buffer RLT Plus (QIAGEN, 1053393), and total RNA was extracted from the lysate using an RNA clean and concentrator-25 kit (Zymo Research, R1018) with the following modification: 752.5 µL of preconditioned RNA binding buffer (367.5 µL of RNA binding buffer (supplied with an RNA clean and concentrator-25 kit), 367.5 µL of absolute EtOH and 17.5 µL of 20% SDS) was added to the cell lysate. After mixing, the material was loaded onto the column of an RNA clean and concentrator-25 kit, and the manufacturer's instructions were followed for the remaining steps.
Rna clean and concentrator 25 kit
Manufactured by Zymo Research
Sourced in United States
The RNA Clean and Concentrator-25 kit is a tool designed to purify and concentrate RNA samples. It employs a silica-based membrane technology to capture and wash RNA, before eluting the purified RNA for further use.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop, by Thermo Fisher Scientific (4 mentions)
Hiscribe t7 high yield rna synthesis kit, by New England Biolabs (3 mentions)
Hiseq 2500, by Illumina (3 mentions)
Turbo dnase, by Thermo Fisher Scientific (3 mentions)
Rlt plus buffer, by Qiagen (2 mentions)
Rneasy plus mini kit, by Qiagen (2 mentions)
Rna clean and concentrator 5, by Zymo Research (2 mentions)
Agilent bioanalyzer, by Agilent Technologies (2 mentions)
Cycloheximide (chx), by Merck Group (2 mentions)
Superscript 3, by Thermo Fisher Scientific (2 mentions)
Illustra microspin s 400 hr column, by GE Healthcare (2 mentions)
E coli poly a polymerase, by New England Biolabs (2 mentions)
Apppg cap, by New England Biolabs (2 mentions)
Rnase free dnase 1, by New England Biolabs (2 mentions)
Hiseq x10, by Illumina (2 mentions)
Monarch pcr and dna cleanup kit, by New England Biolabs (1 mentions)
Mmessage mmachine t7 transcription kit, by Thermo Fisher Scientific (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
Rna 6000 pico kit, by Agilent Technologies (1 mentions)
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (1 mentions)
36 protocols using rna clean and concentrator 25 kit
1
Total RNA Extraction from Mammalian Cells
2
Fabrication of RNA Hairpin for SARS-CoV-2 Studies
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The fabrication of the RNA hairpin has been described in detail in Papini et al., 2019 (link). The RNA hairpin is made of a 499 bp dsRNA stem terminated by a 20 nt loop that is assembled from three ssRNA annealed together, and two handles, one of 856 bp at the 5′-end and one 822 bp at the 3′-end. The handles include either a 343 nt digoxygenin-labeled ssRNA or a 443 nt biotin-labeled ssRNA. Upon applied force above ~21 pN, the hairpin opens and frees a 1043 nt ssRNA template for SARS-CoV-2 replication. To obtain the different parts of the RNA construct, template DNA fragments were amplified via PCR, purified (Monarch PCR and DNA Cleanup Kit) and in vitro transcribed (NEB HiScribe T7 High Yield RNA Synthesis Kit). Transcripts were then treated with Antarctic Phosphatase and T4 Polynucleotide Kinase. RNAs were purified using the RNA Clean and Concentrator-25 kit (Zymo Research). Individual RNA fragments were annealed and ligated with T4 RNA ligase 2 (NEB) to assemble the RNA hairpin.
The template contains 250 U (24%), 253 A (24%), 273 C (26%), and 267 G (26%).
The template contains 250 U (24%), 253 A (24%), 273 C (26%), and 267 G (26%).
3
Ribosome Profiling: Technique and Analysis
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The ribosomal profiling technique was carried out as reported previously49 (link), with a few modifications as described below. To prepare RFs, RNase I and DNA I were added to the lysate and incubate for 45 min at room temperature. Size exclusion columns were equilibrated with polysome buffer by gravity flow. Digested RFs were added to the column and centrifuged. Next, 10 μL 10% SDS was added to the elution, and RFs with a size greater than 17 nt were isolated according to the RNA Clean and Concentrator-25 kit (Zymo Research; R1017). rRNA was removed using a method reported previously50 (link). Briefly, short (50–80 bases) antisense DNA probes complementary to the rRNA sequences were added to a solution containing RFs, and RNase H and DNase I were added to digest the rRNA and residual DNA probes. Finally, RFs were further purified using magnetic beads. After obtaining the ribosome footprints above, Ribo-seq libraries were constructed using the NEBNextR Multiple Small RNA Library Prep Set for Illumina. Briefly, adapters were added to both ends of the RFs, followed by reverse transcription and PCR amplification. The 140–160 bp size PCR products were enriched to enriched to generate to a cDNA library and sequenced using Illumina HiSeq X10 by Gene Denovo Biotechnology Co. (Guangzhou, China).
4
RNA-seq Analysis of Pathogen Induction
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RNA samples were cleaned and concentrated using RNA Clean and Concentrator™ − 25 Kit (ZYMO RESEARCH). The quality of the RNA was evaluated using NanoDrop and Agilent Bioanalyser (RNA Plant Nano, DNA High Sensitivity and RNA Eukaryote Pico Chips). RiboZero libraries were constructed from RNA samples at 0 and 24 h time after inoculation. Then, the libraries were sequenced on an Illumina HiSeq 2500 ultra-high-throughput sequencing system with 150 bp paired-end reads. Since preliminary gene expression analysis of previously described pathogen inducible genes through real time quantitative RT-PCR (qRT-PCR) revealed induction of genes within 24 h after inoculation, the 48 h samples were not included in RNA-seq but used to study expression of individual genes through qRT-PCR.
5
In Vitro Protein Expression Protocol
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Plasmids were linearized using ApaLI for 4 h at 37 °C and purified (QIAquickPCR Purification Kit, Qiagen, UK). One microgram of of DNA was used to transcribe capped and poly-adenylated messenger RNAs using the mMESSAGE mMACHINE T7 Transcription Kit (Thermo Fisher Scientific, UK) according to the manufacturer’s description and purified using the RNA Clean and Concentrator-25 Kit (Zymo Research, USA). RNA integrity was determined via the Bioanalyser, using the RNA 6000 Pico Kit (Agilent, USA). Two micrograms of RNA were used in the Rabbit Reticulocyte Lysate System, Nuclease Treated (Promega, UK) according to the manufacturer’s instructions. Samples were then immunoblotted.
6
CVS-11 Nucleoprotein Gene Amplification
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The complete nucleoprotein gene of challenge virus standard (CVS-11) was amplified using primers Lys001 (5’-ACGCTTAACGAMAAA-3’) and 304 (5’-TTGACAAAATCTTCTCAT-3’) as described previously [33 (link)]. Amplification products were purified using the Zymoclean Gel DNA Recovery Kit (Zymo Research) followed by cloning using the pGEM-T Easy vector system (Promega) according to the manufacturer’s instructions. Recombinant clones were characterized by sequencing using the BigDye Terminator V3.1 cycle sequencing kit (Thermo Fisher Scientific) and an ABI3500xL genetic analyser (Applied Biosystems) to determine orientation. A single recombinant clone containing the insert in the correct orientation with regard to the SP6 promoter was selected, and the insert was in vitro transcribed using the MegaScript kit (Ambion) according to the manufacturer’s instructions. In vitro transcribed RNA was purified using the RNA Clean and Concentrator-25 kit (Zymo Research) and quantified using the Qubit 3.0 fluorometer (Invitrogen). Contamination with plasmid DNA was ruled out with no-RT controls.
7
In vitro Transcribed RNA Production
8
RNA Fragmentation and Ac4C Modification
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Total RNA purification from cancer cells (150 μg) was conducted through an RNA extraction kit, fragmenting these purified samples into nearly 200 nt pieces using 10 × fragmentation buffer (100 mM Tris-HCl, 100 mM ZnCl2 in nuclease-free H2O) followed by incubation for 6 min at 70 °C. Ethylenediaminetetraacetic acid was introduced immediately thereafter to cease the reaction. Our study employed a Zymo RNA clean and concentrator-25 kit (Irvine, CA, USA) for further purification and recovery of the fragmented RNA. Herein, we utilized anti-ac4C (ab252215, Abcam) and control rabbit immunoglobulin G (IgG) antibodies, aiming at interaction with the RNA's ac4C modification sites. RIP was conducted through Pierce™ Protein A/G Magnetic Beads (88,802, Thermo Fisher Scientific, Waltham, MA, USA) per the protocols.
9
FMDV Replicon Plasmid Production
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The construction, linearisation and purification of FMDV replicon plasmids has been described previously [24, 60] . Linear plasmids were transcribed in vitro using the T7 RiboMAX Express Large Scale RNA Production System (Promega) using 'half sized' reactions whereby 250 ng linear plasmid was added to a reaction mixture using half the volume suggested for all reagents in the manufacturer's protocol. Reactions were incubated at 37 °C for 1.5 h prior to addition of 1 µl DNase I and further incubation at 37 °C for 20 min. The resulting RNA was purified using the RNA Clean and Concentrator-25 kit (Zymo Research) prior to quantification by NanoDrop (Thermo Fisher) and confirmation of integrity by denaturing MOPS-formaldehyde gel electrophoresis.
10
Total RNA Extraction and RNA-seq Library Prep
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Total RNA for RNA-seq experiments was extracted from 80% confluent cells. Then, 1 million cells were trypsinized, pelleted, and resuspended in 1 ml TRIreagent (Sigma Aldrich). Total RNA was extracted by phenolchloroform and purified through the RNA Clean and Concentrator-25 kit (Zymo Research), following the manufacturer’s instructions. Ribosomal RNA was depleted and purified using Ribo-Zero rRNARemoval Kit (Illumina) and RNA Clean and Concentrator-5 (Zymo Research). The quality of extracted RNA was checked by Agilent Bioanalyzer on Agilent RNA 6000 Pico Chips (Agilent technologies) both before and after rRNA depletion. RNA-seq libraries were generated using the NEBNext Ultra Directional RNA Library Prep kit for Illumina (NEB). Agilent DNA High Sensitivity Chips (Agilent Technologies) were used to check library size and quality. Samples were sequenced on an Illumina HiSeq 1500 platform in single-end using 50-bp reads. The experiment was done in three independent biological replicates.
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