To prepare any small RNA species with 5′-triphosphate moieties, total RNA was first treated with 1 U Terminator 5′-phosphate-dependent exonuclease (epicenter, TER51020) for 30 min at 30 °C in the supplied buffer. This degrades all RNA with 5′-monophosphates (miRNA, rRNA) and leaves 5′-di/triphosphate and capped RNA intact. RNA was acid phenol:chloroform extracted and ethanol precipitated as before. The Terminator-treated samples were then treated with 5′-polyphosphatase (epicenter, RP8092H) for 30 min at 37 °C to remove the γ-phosphates and β-phosphates, leaving them as 5′-monophosphates. RNA was again extracted by acid phenol-chloroform and ethanol precipitated. These samples were then used for small RNA sequencing library preparation as above.
Rp8092h
Manufactured by Illumina
The RP8092H is a lab equipment product manufactured by Illumina. It is a high-performance instrument designed for specific laboratory applications. The core function of the RP8092H is to perform precise and efficient processing of samples for various research and analysis purposes. No further details can be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (2 mentions)
Truseq small rna sample kit, by Illumina (1 mentions)
Bio 38033, by Meridian Bioscience (1 mentions)
Truseq small rna library, by Illumina (1 mentions)
Bioanalyser high sensitivity, by Agilent Technologies (1 mentions)
Ter51020, by Illumina (1 mentions)
Nextseq, by Illumina (1 mentions)
T4 rna ligase, by New England Biolabs (1 mentions)
Qubit 1x dsdna hs assay kit, by Thermo Fisher Scientific (1 mentions)
Truncated t4 rna ligase, by New England Biolabs (1 mentions)
Superscript 3, by Thermo Fisher Scientific (1 mentions)
Nextseq 500, by Illumina (1 mentions)
Q5 high fidelity dna polymerase, by New England Biolabs (1 mentions)
4 protocols using rp8092h
1
Enriching Small RNA Sequencing Libraries
2
Small RNA Sequencing of Synchronized Worms
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Synchronised animals were grown to 1 day-old adults 20 °C. After being washed with M9 to remove bacteria, animals were resuspended in TRIsure (Bioline, BIO-38033). Animals were lysed with 5× freeze-thaw cycles in liquid nitrogen. Total RNA was isolated by chloroform extraction. For 5′ -independent libraries, 5 µg of total RNA was treated with 5′ polyphosphatase (Epicenter, RP8092H). Small RNAs were indexed using the TruSeq small RNA sample kit (Illumina) and size selected by gel separation in 6% TBE gels (Life Tech) and subsequently purified.
3
Single-Cell RNA Sequencing of Triptolide-Treated Drosophila
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For each condition 20 10ˆ6 of Triptolide treated Drosophila cells were spiked with 2 10ˆ6 untreated mouse embryonic stem cells. scRNA protocol was adapted from (Nechaev et al., 2010 (link)). Cells were re-suspended in ice-cold lysis buffer (10mM Tris (pH = 7.4), 10mM NaCl, 3mM Mgcl2, 0.1mM EDTA, 0.5% NP40), incubated 10min on ice, span down. Nuclei were washed with ice cold (10mM Tris (pH = 7.4), 10mM NaCl, 3mM Mgcl2, 0.1mM EDTA) and nuclear pellets were dissolved in Trizol (Thermofisher). RNA was size selected (17-200bp) using a two-step column purification strategy (RNA clean and concentrator, Zymo-R1016). 10 μg of purified RNA was successively treated by 5′ dephosphorylation - 20U at 37°C for 30min (Epicenter - RP8092H); 5′ terminator exonuclease - 1U in Buffer A at 30°C for 60min (Epicenter - TER51020); cap-clip decapping enzyme – 5U at 37°C for 90min. After each reaction, short RNA was column purified (RNA clean and concentrator, Zymo-R1016). The resulting RNA was used for library preparation using TruSeq small RNA library (Illumina). Libraries were purified on 6% TBE gels (150-300bp – Novex - EC6265BOX). Size distribution of the libraries were controlled on Bioanalyser High sensitivity (Agilent 5067-4626). Two biologically independent inhibition time courses were performed. The samples were run on an Illumina NextSeq generating 38bp paired-end reads.
4
Small RNA Sequencing Library Preparation
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Small RNAs (18 to 30-nt) were size selected on denaturing 15% polyacrylamide gels (Bio-Rad 3450091) from total RNA samples. Small RNAs were treated with 5′ RNA polyphosphatase (Epicenter RP8092H) and ligated to 3′ pre-adenylated adapter with Truncated T4 RNA ligase (NEB M0373L). Small RNAs were then hybridized to the reverse transcription primer, ligated to the 5′ adapter with T4 RNA ligase (NEB M0204L), and reverse transcribed with Superscript III (Thermo Fisher 18080-051). Small RNA libraries were amplified using Q5 High-Fidelity DNA polymerase (NEB M0491L) and size selected on a 10% polyacrylamide gel (Bio-Rad 3450051).
Library concentration was determined using the Qubit 1X dsDNA HS Assay kit (Thermo Fisher Q33231) and quality was assessed using the Agilent BioAnalyzer. Libraries were sequenced on the Illumina NextSeq500 (SE 75-bp reads) platform. For mRNA sequencing, total RNA samples were submitted in triplicate to Novogene Genome Sequencing Company for library preparation. Libraries were sequenced on the Illumina (PE 150-bp reads) platform.
Library concentration was determined using the Qubit 1X dsDNA HS Assay kit (Thermo Fisher Q33231) and quality was assessed using the Agilent BioAnalyzer. Libraries were sequenced on the Illumina NextSeq500 (SE 75-bp reads) platform. For mRNA sequencing, total RNA samples were submitted in triplicate to Novogene Genome Sequencing Company for library preparation. Libraries were sequenced on the Illumina (PE 150-bp reads) platform.
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