RNA yield was determined by Qubit RNA HS assay kit (ThermoFisher Scientific, Reinach, Switzerland). 50 ng of total RNA were subjected to depletion of ribosomal RNA by the use of the RiboMinus™ Eukaryote System v2 kit (ThermoFisher Scientific/Life technologies™/Ambion®, Reinach, Switzerland). As next step, RNA library preparation was achieved by application of the NEXTflex™ Illumina RNA-Seq Library Prep Kit v2 with Molecular Indexes by Bioo Scientific© (Bioo Scientific, Austin, Texas, United States) according to the manufacturer’s recommendations. Library size was assessed before sequencing using the Agilent 2100 Bioanalyzer high sensitivity DNA analysis kit (Agilent, Waldbronn, Germany). Barcoded libraries were sequenced on an Illumina HiSeq 2500 in 75bp paired-end high output mode.
Ribominus eukaryote system v2 kit
Manufactured by Thermo Fisher Scientific
Sourced in Switzerland
The RiboMinus™ Eukaryote System v2 kit is a laboratory tool designed to remove ribosomal RNA (rRNA) from eukaryotic total RNA samples. The kit utilizes a hybridization-based approach to selectively capture and remove rRNA, allowing for the enrichment of non-rRNA species, such as messenger RNA (mRNA) and other RNA transcripts.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (2 mentions)
Hiseq 2500, by Illumina (1 mentions)
Qubit rna hs assay kit, by Thermo Fisher Scientific (1 mentions)
Agilent 2100 bioanalyzer high sensitivity dna analysis kit, by Agilent Technologies (1 mentions)
Rneasy minelute cleanup kit, by Qiagen (1 mentions)
High pure viral nucleic acid extraction kit, by Roche (1 mentions)
Minelute pcr purification kit, by Qiagen (1 mentions)
Superscript vilo cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Nextseq, by Illumina (1 mentions)
Kapa library quantification, by Roche (1 mentions)
Truseq rna sample preparation kit, by Illumina (1 mentions)
4 protocols using ribominus eukaryote system v2 kit
1
Quantifying Eukaryotic Transcriptome via RNA-seq
2
Quantitative Analysis of m6A in RSV RNA
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RSV RNA (250 mg) was extracted from highly purified rgRSV virions using an RNeasy Mini kit (Qiagen) and purified twice with RiboMinus Eukaryote System v2 kit (Thermo Fisher). To examine the purity of virion RNA, oligo d(T) was used for reverse transcription, followed by qPCR for quantification for β-actin and viral N and G mRNAs. Virion RNA which was free of contamination of host RNA and viral mRNAs was used for liquid chromatography-mass spectrometry (LC-MS/MS), m6A antibody pulldown assay, and m6A-seq. Purified RNA was digested and subjected to a quantitative analysis of the m6A level using LC-MS/MS as previously described7 (link).
3
RNA-Seq Library Preparation from Spiked Cells
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Genomic DNA and total RNA were extracted with the High Pure Viral Nucleic Acid extraction kit (Roche) and the RNeasy minikit (Qiagen), respectively. Genomic DNA was quantified using a NanoDrop 2100 spectrophotometer, and concentrations were adjusted to 20 ng/µl before performing high-throughput sequencing. For each spike level and in order to obtain enough starting material, total RNA was extracted from the equivalent of 8 × 106 spiked HeLa cells (final volume of 200 µl). After quantification (NanoDrop 2100), the 200-µl samples were processed through two successive rounds of rRNA depletion using the RiboMinus eukaryote system v2 kit (Ambion - Thermo Fisher Scientific). After concentration and elution in a volume of 15 µl (RNeasy MinElute cleanup kit; Qiagen), 14 µl of the extract was reverse transcribed using the SuperScript VILO cDNA synthesis kit (Invitrogen – Thermo Fisher Scientific). Double-stranded cDNA was synthesized using the NEBNext mRNA second-strand synthesis module kit (New England Biolabs). For each sample, all second-strand reactions were pooled, cleaned, and concentrated using the MinElute PCR purification kit from Qiagen, and concentrations were adjusted to 10 ng/µl before HTS. Except when specified, all kits were used following the manufacturer’s instructions.
4
Ythdc2 RNA Immunoprecipitation Protocol
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Beads and Input samples for two biological replicates of both wild type and Ythdc2-/- were resuspended in PK buffer (10 mM Tris pH 7.0, 100 mM NaCl, 1 mM EDTA, 0.5% SDS), 5 μL Proteinase K and 1 μL RNasOUT (100 μL total volume). Samples were incubated for 1 hr at 42°C and then 1 hr at 55°C. RNA was isolated using TRIzol (Life Technologies), according to manufacturer’s instructions. 1 μL GlycoBlue and 750 μL isopropanol were added to the aqueous phase and incubated at −20°C. Samples were centrifuged for 40 min at max speed at 4°C and washed 2x in 75% cold ethanol. The RNA was resuspended in 10 μL ultrapure water. Ribosomal RNA was removed from the Input and fRIP samples using the RiboMinus Eukaryote System v2 Kit (Ambion) following manufacture’s instructions. Library preparation for high-throughput sequencing was performed using a TruSeq RNA Sample preparation Kit (Illumina, San Diego, CA) per manufacture’s instruction, except RNA was not fragmented with FPF solution. One microliter of each sample was used for quantification with Kapa Library quantification (Kapa Biosystems) and then sent for deep sequencing on the Illumina NextSeq for 2 × 75 bp cycle run.
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