Our modified high-throughput SHAPE-MaP protocol was performed on a Tecan Freedom Evo. We purchased mRNA clones of TPT1 and LCP1 (Origene—SC323772 and SC118739) and designed primers to introduce variants into select regions (Supplemental Table S6 ). Site-directed mutagenesis was performed with an NEB Q5 Site-Directed Mutagenesis Kit, but without customary transformation or cloning. Instead, we PCR-amplified the site-directed target after ligation using primers spanning the entire mRNA; the forward primer included a T7 promoter (NEB Q5 Hotstart). Ampure bead purification was performed to purify the DNA (Beckman Coulter—Ampure XP). Then we performed in vitro transcription with the T7 Polymerase to synthesize RNA (NEB T7 Polymerase). To remove DNA, the sample was treated with TurboDNAse for 15 min at 37°C (ThermoFisher Scientific TurboDNAse Kit). Ampure bead purification was performed to purify the RNA (Beckman Coulter—RNAClean XP). To fold the RNA, samples were incubated at 37°C for 10 min in buffer containing 100 mM Na-HEPES, pH 8.0, 100 mM NaCl, and 10 mM MgCl2. The RNA was incubated for 5 min at 37°C with 10% dimethyl sulfoxide (DMSO) or with 10 mM 1-methyl-7-nitroisatoic anhydride (1M7) in DMSO. Ampure bead purification was performed to purify the modified RNA (Beckman Coulter—RNAClean XP).
Rnaclean xp
Manufactured by Beckman Coulter
Sourced in United States
RNAClean XP is a magnetic bead-based system for the purification of RNA from various sample types. The core function of this product is to efficiently isolate and concentrate RNA molecules from complex biological samples, providing high-quality RNA for downstream applications such as qRT-PCR, Northern blotting, and RNA sequencing.
Automatically generated - may contain errors
Lab products found in correlation
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (4 mentions)
Ribo zero rrna removal kit, by Illumina (3 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Turbo dnase, by Thermo Fisher Scientific (3 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (3 mentions)
Hiscribe t7 high yield rna synthesis kit, by New England Biolabs (3 mentions)
Maxima h reverse transcriptase, by Thermo Fisher Scientific (3 mentions)
Tcl buffer, by Qiagen (3 mentions)
Nuclease free water, by Qiagen (3 mentions)
Facsaria 2, by BD (3 mentions)
Ampure xp, by Beckman Coulter (3 mentions)
Fast sybr green master mix, by Thermo Fisher Scientific (2 mentions)
Truseq rna sample prep kits v2, by Illumina (2 mentions)
Rneasy column, by Qiagen (2 mentions)
Dnase, by Qiagen (2 mentions)
Improm 2, by Promega (2 mentions)
Hiseq 2500 system, by Illumina (2 mentions)
T7 rna polymerase ver 2, by Takara Bio (2 mentions)
Rabbit reticulocyte lysate translation system, by Promega (2 mentions)
Rnase a, by Promega (2 mentions)
39 protocols using rnaclean xp
1
High-throughput SHAPE-MaP protocol
2
RNA Isolation, cDNA Synthesis, and RNA-Seq Library Preparation
3
Comprehensive RNA Extraction and Analysis
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Total RNA was purified onto RNeasy columns (Qiagen) and treated on-column with DNase (Qiagen). Complementary DNA (cDNA) was produced using the reverse transcriptase ImPromII (Promega). 10 ng of cDNA were used for Real-time RT-PCR reactions with FAST SYBR Green Master Mix (Applied Biosystems).
For RNA-Seq, total RNA from 107 cells was purified using Trizol (Invitrogen), treated with Turbo DNase (Ambion) and purified with RNA Clean XP (Agencourt). 5 μg of purified RNA were then treated with Ribozero rRNA removal kit (Epicentre) and ethanol precipitated. RNA quality and removal of rRNA were checked with the Agilent 2100 Bioanalyzer (Agilent Technologies). Libraries for RNA-Seq were then prepared with the TruSeq RNA Sample Prep Kits v2 (Illumina) following manufacturer instructions starting from the RNA fragmentation step.
For RNA-Seq, total RNA from 107 cells was purified using Trizol (Invitrogen), treated with Turbo DNase (Ambion) and purified with RNA Clean XP (Agencourt). 5 μg of purified RNA were then treated with Ribozero rRNA removal kit (Epicentre) and ethanol precipitated. RNA quality and removal of rRNA were checked with the Agilent 2100 Bioanalyzer (Agilent Technologies). Libraries for RNA-Seq were then prepared with the TruSeq RNA Sample Prep Kits v2 (Illumina) following manufacturer instructions starting from the RNA fragmentation step.
4
Circular RNA Expression Profiling in Major Depressive Disorder
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To investigate the expression profile of circRNAs in MDD, three MDD and three matched healthy controls were analyzed by HTS. First, the double-stranded and single-stranded DNA present in RNA samples were removed by DNAse. The Ribo-Zero rRNA Removal Kit (Illumina, Inc.) and RNase R (Epicenter, lnc) were utilized to remove ribosomal RNA and linear RNA, respectively. Furthermore, purification was carried out using Agencourt RNAClean XP magnetic beads. All operations were performed according to the manufacturer’s protocols. Finally, sequencing was performed on the Hiseq 4000 or Hiseq X-ten platform (BGI-Shenzhen, China).
5
Tn5 Tagmentation of RNA/DNA Hybrids
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For testing tagmentation activity of Tn5 on RNA/DNA hybrids, reactions were carried out as above, with 25ng mRNA derived RT products as substrate. The tagmentation products were then purified using 1.8X Agencourt RNAClean XP
6
Quantitative PCR Analysis of Immune Cell Markers
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Total RNA was isolated from sorted cells using RNAClean XP (Beckman Coulter), and cDNA was synthesized with SuperScript IV Reverse Transcriptase (Invitrogen). Quantitative PCR reactions were established using QuantiFast Multiplex PCR Kits (QIAGEN) and run on QuantStudio 7 (Applied Biosystems). The following primers were used: GATA Binding Protein 3 (Gata3; Mm00484683), inhibitor of DNA binding 2 (Id2; Mm00711781_m1), inhibitor of DNA binding 3 (Id3; Mm00492575_m1), interleukin-5 (Il5; Mm00439646_m1), interleukin-10 (Il10; Mm00439616_m1), interleukin-13 (Il13; Mm00434204_m1), interleukin-33 (Il33; Mm00505403_m1), plexin A1 (Plxna1; Mm00501110_m1), plexin A4 (Plxna4; Mm01163292_m1), RAR-related orphan receptor A (Rora; Mm01173766_m1), semaphorin 6d (Sema6d; Mm00553142_m1), and endogenous control gene Actb (4352341E, Applied Biosystems). The gene expression data were normalized by the expression of an endogenous control gene.
7
Efficient RNA Purification at Varying pH
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Example 2
RNA was purified using the above exemplary protocol with binding buffers B1 at varying pH (4.3 and 6.3) and varying RNA concentrations (20 μg RNA dissolved in 20 μl or in 200 μg solution). The same sample was also purified using the Agencourt® RNAClean XP kit by Beckman Coulter. The relative total RNA yield for each method was quantified and is presented in
8
Binding Affinity Measurements of Crc and Hfq Proteins
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DNA templates that carried the wild or mutated crcZ (339 bp), crcY (315 bp), oprB (1254 bp), gltR (761 bp), zwf (1486 bp), citN (1344 bp), benR (984 bp), and nifH (882 bp) were synthesized by Sangon Biotech Co. All the synthesized DNA templates were sequenced to assure the absence of undesired mutations. For in vitro transcription from synthesized templates, the T7 High Yield RNA Transcription Kit (Vazyme) was used according to the manufacturer’s instructions. The resulting ssRNA oligonucleotides were purified by the RNAClean XP (Beckman Coulter Inc.) according to the manufacturer’s instructions. The purified Crc and Hfq proteins were labelled with a Monolith NTTM Protein labelling kit RED-NHS according to the manufacturer’s instructions (No. MO-L011, NanoTemper Technologies). For the measurements, the concentration of the labelled Hfq (20 nM) or Crc (200 nM) proteins was kept constant, while the concentrations of non-labelled ssRNA oligonucleotides varied from 0.3 μM to 10 μM. The dissociation constants (Kd) were calculated.91 (link) Data analyses were performed with NanoTemper Analysis software (NanoTemper Technologies).
9
Synthetic HDV RNA Generation and Extraction
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The target gene fragments of HDV were synthesized and cloned into the pUC57 vector (Biomed Biotechnology, Beijing China). Synthetic DNA targets with a forward primer that contained a T7 promoter sequence were transcribed in vitro to generate synthetic RNA targets. The product was used as the template using the HiScribe T7 High Yield RNA Synthesis Kit (New England Biolabs, USA) at 37°C overnight.
The transcribed RNA was then treated with RNase-free DNase I (Qiagen, Germany) to remove any remaining DNA according to the manufacturer’s instructions. RNA was purified using RNA Clean XP (Beckman Coulter, USA), quantified by Nanodrop and Qubit and diluted in nuclease-free water to working concentrations. HDV RNA was extracted from 200 µl plasma samples using an automated nucleic acid extraction system (Bioteke, China) according to the manufacturer’s instructions.
According to previous studies, thermal shock was used to disrupt the secondary structure of HDV RNA, that is, 10 µL of HDV RNA at 95°C for 10 min, immediately followed by cooling to −80°C [22 (link),23 (link)].
The transcribed RNA was then treated with RNase-free DNase I (Qiagen, Germany) to remove any remaining DNA according to the manufacturer’s instructions. RNA was purified using RNA Clean XP (Beckman Coulter, USA), quantified by Nanodrop and Qubit and diluted in nuclease-free water to working concentrations. HDV RNA was extracted from 200 µl plasma samples using an automated nucleic acid extraction system (Bioteke, China) according to the manufacturer’s instructions.
According to previous studies, thermal shock was used to disrupt the secondary structure of HDV RNA, that is, 10 µL of HDV RNA at 95°C for 10 min, immediately followed by cooling to −80°C [22 (link),23 (link)].
10
Primer Pair Validation for eRPA Assays
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Four N gene forward primers (JQ217, CCMS041, CCMS047, and CCMS051) were paired with the reverse primer JQ224. These four primer pairs as well as JQ217 + JQ223 were used in eRPA assays with a water-only sample input. eRPA assays were incubated at 42 °C for 10 min. Amplification products were cleaned up using RNA Clean XP (Beckman Coulter) at 2.5× concentration and eluted in 20 µL of nuclease-free water. Purified products were cloned using the Zero Blunt TOPO PCR Cloning Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions and Sanger sequenced. The identity of cloned products was determined by first aligning the sequences to the vector sequence using Samtools (v1.9) with an allowed multimapping of k = 10,000. The sam files were then visualized in IGV (v2.6.2) where the direction, sequence, and copy number of primer oligomers were manually annotated. In Table S2 , the direction of the primer indicates a forward or reverse direction with respect to the vector, while the space sequence is the string of nucleotides between two primers. Overlap indicates the primers were overlapping with respect to the vector, “*” indicates there was no space between the primers, and listed nucleotides indicate the sequence between two primers.
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