RNA from EVs was extracted using ExoQuick Exosome RNA Column Purification Kit (EQ808A-1, System Biosciences, CA, USA) and miRNA library synthesis was performed using the QIAseq miRNA Library Kit (QIAGEN, Germany) following manufacturer instructions (QIAseq miRNA Library Kit Handbook ver. 03/2020). Prior to sequencing, the libraries underwent electrophoretic control on DNA1000 capillary electrophoresis cartridges of Bioanalyzer 2100 (Agilent Technologies, Italia), and quantification by Qubit fluorimetric DNA High Sensitivity Assay (Thermo Fisher Scientific, USA). The libraries have been sequenced in 76 bp Single End run on a NextSeq 500 platform (Illumina, USA, RRID:SCR_017958) with automated trimming and demultiplexing made online to the sequencing with the Illumina Cloud BaseSpace Sequencing Hub.
Qiaseq mirna library kit
Manufactured by Qiagen
Sourced in Germany, United States, Netherlands, United Kingdom, Italy
The QIAseq miRNA Library Kit is a laboratory equipment product designed for the preparation of small RNA sequencing libraries. It enables the efficient and sensitive capture of miRNA and other small RNA molecules from various sample types.
Automatically generated - may contain errors
Lab products found in correlation
Agilent 2100 bioanalyzer, by Agilent Technologies (35 mentions)
Mirneasy mini kit, by Qiagen (26 mentions)
2100 bioanalyzer, by Agilent Technologies (22 mentions)
Novaseq 6000, by Illumina (17 mentions)
Truseq pe cluster kit v3 cbot hs, by Illumina (16 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (16 mentions)
Nextseq 500, by Illumina (13 mentions)
Hiseq 2500, by Illumina (11 mentions)
Hiseq platform, by Illumina (11 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (11 mentions)
Mirneasy serum plasma kit, by Qiagen (9 mentions)
Nextseq 550, by Illumina (8 mentions)
Nextseq 500 system, by Illumina (8 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (8 mentions)
Novaseq 6000 platform, by Illumina (7 mentions)
High sensitivity dna kit, by Agilent Technologies (6 mentions)
Nextseq 550 system, by Illumina (5 mentions)
Mirneasy micro kit, by Qiagen (5 mentions)
Mirneasy serum plasma advanced kit, by Qiagen (5 mentions)
Novaseq 6000 system, by Illumina (5 mentions)
215 protocols using qiaseq mirna library kit
1
EV miRNA Extraction and Sequencing
2
Optimized Small RNA Sequencing Library Preparation
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Small RNA libraries from serum samples were obtained using QIAseq® miRNA Library kit (Qiagen, Hilden, Germany), according to the manufacturer protocol. NGS Library Quality Control (QC) analysis and quantification were performed before sequencing: a) High sensitivity DNA electrophoresis by LabChip GX Touch Nucleic Acid Analyzer (PerkinElmer, Massachusetts, USA) using HT DNA 5K/RNA LABCHIP kit (D-MARK Biosciences, Toronto, Canada) according to the manufacturer’s instructions. We obtained typical electropherograms from small RNA libraries that show a peak between 170-180 bp corresponding to miRNA-sized library; b) quantitative polymerase chain reaction (qPCR) according to the manufacturer’s protocol, using three different primers provided by QIAseq® miRNA Library kit (Qiagen): the first, called NGS 3C Primer, for assessing the performance of 3’ adapter ligation; the second, NGS 5C Primer, for assessing the performance of 5’ adapter ligation and the third, NGS RTC Primer, for the performance of reverse transcription reaction. We obtained a value of threshold cycle (CT) less than 28 indicating all these steps were performed correctly. NGS library concentration was determined by Qubit dsDNA HS assay (Thermo Fisher Scientific) by Qubit® 4.0 Fluorimeter, according to the manufacturer’s protocol.
3
Optimized miRNA Library Prep for NGS
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Library preparation was performed using the QIAseq miRNA Library Kit (cat no: 331505, Qiagen) following the QIAseq miRNA Library Kit Handbook (Qiagen, 2018). Implemented was a prolonged three-ligation step to 18 hours, due to previous experiments.
The kit operates with unique molecular identifiers (UMIs), unique sequences that are integrated into the reverse transcription. Consequently, each miRNA is assigned to a unique sequence, enabling the exact quantification of input miRNA by next-generation sequencing (NGS). This approach reduces the usual amplification bias during polymerase chain reaction.
Quality and quantity control of the libraries was conducted using an automated electrophoresis (Agilent 2100 Bioanalyzer, DNA 1000 Kit), following the corresponding protocol (Agilent Technologies, 2016). NGS was carried out using an illumina sequencing platform (NextSeq500) with a single read length of 75 bp and around 400 million reads per run.
The kit operates with unique molecular identifiers (UMIs), unique sequences that are integrated into the reverse transcription. Consequently, each miRNA is assigned to a unique sequence, enabling the exact quantification of input miRNA by next-generation sequencing (NGS). This approach reduces the usual amplification bias during polymerase chain reaction.
Quality and quantity control of the libraries was conducted using an automated electrophoresis (Agilent 2100 Bioanalyzer, DNA 1000 Kit), following the corresponding protocol (Agilent Technologies, 2016). NGS was carried out using an illumina sequencing platform (NextSeq
4
Comprehensive miRNA Library Preparation
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Libraries were prepared from 5 μl of miRNeasy RNA using NEXTflex® Small RNA Sequencing Kit v3 for Illumina® Platforms (Bioo Scientific) and QIAseq miRNA Library Kit (QIAGEN) and from 2 μl miRNeasy RNA using CleanTag™ Small RNA Library Prep Kit (TriLink), following each of the manufacturers’ instructions. Additionally, libraries were prepared from 5 μl MagnaZol RNA using NEXTflex® Small RNA Sequencing Kit v3 for Illumina® Platforms (Bioo Scientific) and QIAseq miRNA Library Kit (QIAGEN). Library concentrations were measured using Qubit™ dsDNA HS Assay Kit (Thermo Fisher). Quality and concentration of libraries were determined by Fragment Analyzer (Advanced Analytical). Libraries were sequenced on a NextSeq 500 System (Illumina).
5
sRNA Library Preparation and Sequencing
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Starting total template CUSA-EV RNA (4 ng) was used to prepare the small RNA (sRNA) libraries using a QIASeqTM miRNA Library Kit (Qiagen, Venlo, Netherlands) [104 (link)]. The sRNA library was prepared, quality control and NGS was performed by the Ramaciotti Centre for Genomics, University of New South Wales. NGS was performed on an Illumina® NextSeqTM 500 NGS System (Illumina, San Diego, CA, USA). The libraries were sequenced in a single-end 75 bp, high-output protocol to generate an output of up to 400 million reads at a Q30 > 85%.
6
miRNA Sequencing Library Preparation
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We generated miRNA libraries for deep sequencing as QIAseqTM miRNA Library Kit (QIAGEN, Germantown, MD, USA) protocol described using 200ng of total RNA. This process included following steps: (1) 3′ adaptor ligation; (2) 5′ adaptor ligation; (3) cDNA synthesis; (4) PCR amplification; (5) libraries qualification using an Agilent 2100 Bioanalyzer (Agilent Technologies Inc., Wilmington, DE, U.S.A.). The libraries were sequenced by Illumina Hiseq2500 SE50 following the vendor’s recommended protocol. Data processing followed the established procedures. Briefly, the raw reads were subjected to Barcode (demultiplex). The dataset was further processed with cutadapt (version 1.15) to remove adapter dimers, junk, low complexity, common RNA families (rRNA, tRNA, snRNA, snoRNA), and repeats. Subsequently, unique sequences>17 nt in length were mapped to mature species in miRbase (Release 22.1) by FANSe3 to identify known miRNAs and novel 3p- and 5p- derived miRNAs. Length variation at both 3′ and 5′ ends and one mismatch inside of the sequence were allowed in the alignment.
7
miRNA Sequencing Library Preparation
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The miRNA sequencing library was prepared using the QIASeqTM miRNA Library Kit (Qiagen). Total RNA was used as the starting material. A preadenylated DNA adapter was ligated to the 3′ ends of miRNAs, followed by ligation of an RNA adapter to the 5′ end. A reverse-transcription primer containing an integrated Unique Molecular Index (UMI) was used to convert the 3′/5′-ligated miRNAs into cDNA. After cDNA cleanup, indexed sequencing libraries were generated via sample indexing during library amplification, followed by library cleanup. Libraries were sequenced on a NextSeq 500 (Illumina, San Diego, CA, United States) with a 1 × 75 bp read length and an average sequencing depth of ∼10 M reads/sample.
8
Global miRNA Expression Profiling by Microarray
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After confirming RNA quality (RNA integrity number (RIN) > 7) using 2100 Bioanalyzer, samples were processed for global human miRNA microarray profiling. This analysis provides expression data of all known human miRNAs and their isotypes (~2500). RNA integrity was analyzed on the 2100 Bioanalyzer using the Agilent RNA 6000 Pico or Nano Kit (Agilent Technologies, Santa Clara, CA, USA), and RNA was quantified using the Qubit RNA HS Assay Kit (ThermoFisher Scientific, Waltham, MA, USA). The QIASeqTM miRNA Library Kit (Qiagen, Hilden, Germany) was used to prepare miRNA sequencing libraries. Final library concentrations were measured using the Qubit 1X dsDNA HS Assay kit. Library fragment size was determined on the 2100 Bioanalyzer or Agilent 4200 TapeStation (Agilent Technologies), and then libraries were sequenced on a NovaSeq 6000 (Illumina, San Diego, CA, USA) at an average sequencing depth of ~10 M reads/sample and 1 × 75 bp sequencing.
9
Quantitative miRNA Sequencing
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Aliquots of the same RNA samples used for the mRNA-seq analysis described above were analyzed by miRNA-seq. Indexed sequencing libraries were prepared from 200 ng total RNA using the QIAseq miRNA Library Kit (Qiagen) in the GESR according to the manufacturer's instructions. Sequencing was performed on the NextSeq 550 System (Illumina) using 1×75 bp paired-end (SE75) read mode. The miRNA-seq data generated were then analyzed using the QIAseq miRNA quanti cation data analysis software. After quality assessment of the samples, FASTQ les were uploaded to the data analysis center at Qiagen for further analysis. In the rst step, the unique molecular index (UMI) counts were calculated and primary miRNA mapping was performed. In the second step, the UMI counts were analyzed to calculate the changes in miRNA expression. The quanti ed data were then normalized using TMM method.
10
Small RNA Sequencing Library Preparation
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Small RNA libraries were prepared from 150 ng of total RNA, essentially using Qiaseq miRNA library kit (Qiagen Manchester Ltd., Manchester, UK) as previously described [41 (link)]. The kit not only allows the detection of miRNAs, but also other small RNAs, including piRNAs. In our data, most of the results consisted of deregulated miRNAs, with a minor proportion of piRNAs. Thus, the “miRNA expression” reported here, denotes an expression of both miRNAs and piRNAs. Small RNA libraries were validated on a Fragment Analyzer (Agilent Technologies Inc., Santa Clara, CA, USA) with a high sensitivity NGS kit. The sequencing of sRNA libraries was performed on MiSeq system (Illumina, Inc., San Diego, CA, USA) using MiSeq Reagent Kit V3 (Illumina, Inc., San Diego, CA, USA).
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