The quantity and quality of the purified total RNA was assessed using a Thermo Fisher Scientific Qubit 3.0 fluorometer with the Qubit RNA BR Assay Kit (Cat # Q10211, Thermo Fisher Scientific) and an Advanced Analytical Fragment Analyzer System using a Fragment Analyzer RNA Kit (Cat # DNF-471, Agilent Technologies, Santa Clara, USA), respectively. Sequencing libraries were constructed using an Illumina TruSeq Stranded mRNA Library Prep kit (20020595, Illumina, San Diego, USA) in combination with TruSeq RNA UD Indexes (Cat # 20022371, Illumina) according to Illumina’s guidelines. The cDNA libraries were evaluated using a Thermo Fisher Scientific Qubit 3.0 fluorometer with the Qubit dsDNA HS Assay Kit (Cat # Q32854, Thermo Fisher Scientific) and an Agilent Fragment Analyzer (Agilent) with an HS NGS Fragment Kit (Cat # DNF-474, Agilent), respectively. Pooled cDNA libraries were sequenced 100 bp single-end on one lane of an Illumina HiSeq 3000 instrument (Illumina). All base call files were demultiplexed and converted into FASTQ files using Illumina bcl2fastq conversion software. The quality control assessments, generation of libraries, and sequencing were conducted by the Next Generation Sequencing Platform, University of Bern.
Fragment analyzer rna kit
Manufactured by Agilent Technologies
Sourced in United States
The Fragment Analyzer RNA Kit is a laboratory equipment product designed for the analysis and quantification of RNA samples. It provides a reliable and efficient method for assessing the quality and size distribution of RNA molecules within a sample.
Automatically generated - may contain errors
Lab products found in correlation
Qubit rna br assay kit, by Thermo Fisher Scientific (4 mentions)
Qubit 4.0 fluorometer, by Thermo Fisher Scientific (2 mentions)
Agilent fragment analyzer, by Agilent Technologies (1 mentions)
Hs ngs fragment kit, by Agilent Technologies (1 mentions)
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (1 mentions)
Truseq rna ud indexes, by Illumina (1 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (1 mentions)
Truseq stranded mrna library prep kit, by Illumina (1 mentions)
Bcl2fastq conversion software, by Illumina (1 mentions)
Hiseq 3000 instrument, by Illumina (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Nextseq 500, by Illumina (1 mentions)
Nebnext ultra directional rna library prep kit, by New England Biolabs (1 mentions)
Nebnext rrna depletion kit, by New England Biolabs (1 mentions)
Nextseq 2000, by Illumina (1 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
Bioanalyzer high sensitivity dna kit, by Agilent Technologies (1 mentions)
Bioanalyzer 2100 rna 6000 nano kit, by Agilent Technologies (1 mentions)
Rnase free dnase set, by Qiagen (1 mentions)
Rnalater, by Merck Group (1 mentions)
5 protocols using fragment analyzer rna kit
1
RNA Sequencing Library Preparation
2
RNA Extraction and Quality Assessment
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Total RNA was extracted with the RNeasy mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The quantity and quality of the isolated RNA was assessed using a Thermo Fisher Scientific Qubit 4.0 fluorometer with the Qubit RNA BR Assay Kit (Q10211, Thermo Fisher Scientific, Reinach, Switzerland) and an Advanced Analytical Fragment Analyzer System with a Fragment Analyzer RNA Kit (DNF-471, Agilent, Basel, Switzerland). The RNA was also tested by spectrophotometry using a Denovix DS-11 FX (M/F) spectrophotometer/fluorometer to assess the purity of the RNA (DeNovix, Wilmington, DE, USA). The median RIN for the four samples was 9.85 (IQR 0.13). The two highest quality samples (RIN ≥ 9.9), representing one asthmatic and one control horse, were equally pooled based on their RNA concentration.
3
High-Quality RNA-Seq Library Preparation
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Total RNA was extracted from 5 × 106 cells using RNeasy Plus Mini Kit (Qiagen, 74204) according to the manufacturer’s protocol, and gDNA was eliminated by treatment with the RNase-Free DNase Set (Qiagen, 79254). Quality of RNA was assessed using the 2100 Bioanalyzer RNA 6000 Nano kit (Agilent) or the Fragment Analyzer RNA kit (Agilent), all samples had RNA integrity number (RIN) > 8. Total RNA (500 ng) from each sample was depleted of rRNA using the NEBNext rRNA Depletion kit (NEB, E7405L). Strand-specific RNA libraries were prepared using the NEBNext Ultra Directional RNA Library Prep kit (NEB, E7765s), assessed on the Bioanalyzer High Sensitivity DNA kit (Agilent) or the Fragment Analyzer HS NGS kit to ensure good quality and sequenced paired-end on a NextSeq500 (76 bp) or NextSeq 2000 (100 bp; Illumina) in 2–6 biological replicates. All samples are detailed in Supplementary Table 1 . RNA-seq data processing and analysis are detailed in the Supplementary Methods .
4
Bone Tissue RNA Extraction and Purification
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For RNA extraction and library preparation, the tissue between the two inner central screws containing the remaining implant material and newly formed bone tissue was dissected, placed in RNALater® (Sigma-Aldrich), and stored at −20 °C. For total RNA extraction, samples were transferred into microtubes containing 1 ml TRIzol™ Reagent (Invitrogen™) and metal beads for tissue disruption in a benchtop tissue homogenizer (TissueLyser 2, Qiagen, Hilden, DE).
RNA was isolated according to the manufacturer's instructions using the NucleoSpin® RNA Plus Kit (Macherey-Nagel, Oensingen, CH), and genomic DNA was removed by digestion with the provided DNase. The quantity and quality of the purified total RNA was assessed using a Thermo Fisher Scientific Qubit 4.0 fluorometer with the Qubit RNA BR Assay Kit (Q10211, Thermo Fisher Scientific, Waltham, Massachusetts, USA) and an Advanced Analytical Fragment Analyzer System using a Fragment Analyzer RNA Kit (DNF-471, Agilent Technologies, Santa Clara, California, USA), respectively.
RNA was isolated according to the manufacturer's instructions using the NucleoSpin® RNA Plus Kit (Macherey-Nagel, Oensingen, CH), and genomic DNA was removed by digestion with the provided DNase. The quantity and quality of the purified total RNA was assessed using a Thermo Fisher Scientific Qubit 4.0 fluorometer with the Qubit RNA BR Assay Kit (Q10211, Thermo Fisher Scientific, Waltham, Massachusetts, USA) and an Advanced Analytical Fragment Analyzer System using a Fragment Analyzer RNA Kit (DNF-471, Agilent Technologies, Santa Clara, California, USA), respectively.
5
PacBio Iso-Seq for Transcriptome Analysis
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The quantity and quality of the extracted RNA was assessed using a Thermo Fisher Scientific fluorometer (Qubit 4.0) with the Qubit RNA BR Assay Kit (Thermo Fisher Scientific, Q10211) and an Advanced Analytical Fragment
Analyzer System using a Fragment Analyzer RNA Kit (Agilent, DNF-471),
respectively. The RNA was also tested by spectrophotometry using a Denovix DS-11 FX Spectrophotometer / Fluorometer to assess the purity of the RNA. Once all quality control tests confirmed high quality RNA, the "Procedure & Checklist -Iso-Seq Express Template Preparation for Sequel and Sequel II Systems" was followed (PN 101-763-800 Version 02 (October 2019)). Two libraries were created from the same total RNA input (300 ng):
one targeting 2KB transcripts and one targeting >3Kb transcripts. Both libraries were SMRT sequenced using a Sequel binding plate 3.0, sequel sequencing plate 3.0 with a 20h movie time on a PacBio Sequel system on their own SMRT cell 1M v3 LR. The 2Kb library was loaded at 3pM and generated 33.90 Gb and 409'187 ≥ Q20 Circular consensus reads (CCS).
While the >3Kb library was loaded at 3pM and produced 27.04 Gb and 248'304 ≥Q20 CCS. All steps post RNA extraction were performed at the Next Generation Sequencing Platform, University of Bern.
Analyzer System using a Fragment Analyzer RNA Kit (Agilent, DNF-471),
respectively. The RNA was also tested by spectrophotometry using a Denovix DS-11 FX Spectrophotometer / Fluorometer to assess the purity of the RNA. Once all quality control tests confirmed high quality RNA, the "Procedure & Checklist -Iso-Seq Express Template Preparation for Sequel and Sequel II Systems" was followed (PN 101-763-800 Version 02 (October 2019)). Two libraries were created from the same total RNA input (300 ng):
one targeting 2KB transcripts and one targeting >3Kb transcripts. Both libraries were SMRT sequenced using a Sequel binding plate 3.0, sequel sequencing plate 3.0 with a 20h movie time on a PacBio Sequel system on their own SMRT cell 1M v3 LR. The 2Kb library was loaded at 3pM and generated 33.90 Gb and 409'187 ≥ Q20 Circular consensus reads (CCS).
While the >3Kb library was loaded at 3pM and produced 27.04 Gb and 248'304 ≥Q20 CCS. All steps post RNA extraction were performed at the Next Generation Sequencing Platform, University of Bern.
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