RNA was isolated from samples using the NucleoSpin TriPrep kit (MACHEREY‐NAGEL), according to the manufacturer's instructions. RNA integrity was measured using an Agilent 2200 TapeStation and RNA Screen Tapes (Agilent Technologies). Sequencing libraries were prepared using an NEB Next Ultra II RNA Library Kit for Illumina (New England Biolabs), according to the manufacturer's protocol, and prepared libraries were run on an Illumina HiSeq × high‐throughput sequencing system. Paired‐end reads were aligned to the hg19 human genome assembly using STAR.18 Fusion transcripts were detected using Genomon version 2.6.2 (https://github.com/Genomon‐Project/ ) and filtered by excluding fusions: (a) mapping to repetitive regions, (b) with fewer than four spanning reads, (c) that occurred out of frame, or (d) that had junctions not located at known exon–intron boundaries. The expression level of each RefSeq gene was calculated from mapped read counts using HTSeq and normalized using the Bioconductor package, DESeq2 version 1.28.1.23. Supercomputing resources were provided by the Human Genome Center, Institute of Medical Science, The University of Tokyo. Hierarchical clustering was applied and performed using Ward's method to calculate Euclidian distances. Cluster stability was ascertained by consensus clustering using the R package, ConsensusClusterPlus, with 500 iterations.
Rna screen tapes
Manufactured by Agilent Technologies
Sourced in United States, Germany
The RNA Screen Tapes are a specialized laboratory equipment designed for the analysis and size separation of RNA samples. They provide a reliable and efficient way to assess the quality and integrity of RNA samples prior to further downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
2200 tapestation system, by Agilent Technologies (3 mentions)
Agilent 2200 tapestation system, by Agilent Technologies (3 mentions)
Agilent 2200 tapestation, by Agilent Technologies (2 mentions)
Nextseq 500 next generation sequencer, by Illumina (2 mentions)
Agilent 4200 tapestation system, by Agilent Technologies (2 mentions)
Rneasy kit, by Qiagen (2 mentions)
Turbo dna free kit, by Thermo Fisher Scientific (2 mentions)
0.1 mm silica beads, by MP Biomedicals (2 mentions)
Tapestation, by Agilent Technologies (2 mentions)
Rneasy ffpe kit, by Qiagen (2 mentions)
Rneasy plus mini kit, by Qiagen (2 mentions)
Nucleospin triprep kit, by Macherey-Nagel (1 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
2200 tapestation instrument, by Agilent Technologies (1 mentions)
Recoverall total nucleic acid isolation kit, by Thermo Fisher Scientific (1 mentions)
Qubit rna high sensitivity assay kit, by Thermo Fisher Scientific (1 mentions)
4150 tapestation system, by Agilent Technologies (1 mentions)
α amylase, by Merck Group (1 mentions)
Rna clean concentrator 5 kit, by Zymo Research (1 mentions)
Spectrum plant total rna kit, by Merck Group (1 mentions)
16 protocols using rna screen tapes
1
Comprehensive RNA-seq Analysis Pipeline
2
RNA Integrity Assessment Protocol
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RNA concentrations were determined by OD 260 nm measurements on a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE). RNA integrity was assessed by the RNA Integrity Number (RIN) using a 2200 TapeStation Instrument with RNA ScreenTapes (Agilent Technologies, Santa Clara, CA). The RIN is a number on a scale from 1 to 10, where RIN = 10 indicates intact RNA and RIN = 1 indicates completely degraded RNA (Schroeder et al., 2006 (link)). Each TapeStation lane image were scaled separately with the TapeStation Analysis software option “Scale to Sample”.
3
FFPE RNA Extraction and Characterization
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RNA was extracted from 10 tissue sections of 5 μm obtained from representative paraffin blocks. The Recover All Total Nucleic Acid Isolation Kit was used for formalin-fixed paraffin-embedded (FFPE) tissue (Invitrogen) following the manufacturer’s instructions. Quantification of RNA was measured fluorometrically with the Qubit RNA high-sensitivity assay kit (Invitrogen). RNA quality was assessed using RNA Screen Tapes on a 2200 TapeStation system (Agilent, Santa Clara, CA, USA).
4
Oat Grain RNA Extraction Optimization
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Developing grains were first collected from fully emerged panicles at 5 DPE and then at 5 d intervals from 5 to 40 DPE. For each of 3 biological replicates, 10 individual grains (glumes removed) from a single panicle were pooled into a 2-mL tube and snap-frozen in liquid nitrogen. Frozen oat grains (100 mg/sample) were ground to fine powder using a plastic pestle. The RNA was extracted using the Spectrum Plant Total RNA Kit (Sigma-Aldrich) with some additional optimization steps. Samples were pretreated with 0.03% v/v α-amylase (Sigma-Aldrich, A4551-100MG) in lysis buffer, vortexed for 1 min, and incubated at RT for 6 min. β-Mercaptoethanol was then added to 0.01% v/v and samples vortexed again for 30 s prior to centrifugation at RT for 5 min at ∼16,000 × g. The supernatant was then transferred to a filtering column, and the Spectrum Plant Total RNA protocol was followed using the manufacturer's instructions including the on-column DNase treatment. Protocol A was used for the binding buffer solution. Samples were further purified using RNA Clean & Concentrator-5 kit (Zymo Research, R1016) to ensure sufficient RNA purity and concentration. RNA analyses were undertaken using a 4150 TapeStation System (Agilent) with RNA Screen Tapes (Agilent, 5067-5576) following the manufacturer's instructions.
5
Neutrophil and Macrophage RNA Sequencing
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The sequencing workflow was performed as previously described51 (link). Briefly, RNA from sorted neutrophils and macrophages (purity 99–100%) was extracted with an RNeasy Plus Micro Kit (QIAGEN) according to the manufacturer’s protocol. Extracted RNA quality and quantity were assessed using an Agilent 2200 TapeStation System (Agilent) with RNA ScreenTapes (Agilent). Next-generation sequencing libraries were created with 10 ng of RNA from samples with distinct 18S and 28S peaks and RNA integrity number values greater than 7, using an NEBNext Ultra II Directional RNA Library Prep Kit from Illumina (New England Biolabs) according to the manufacturer’s protocol. RNA libraries were pooled and sent for single-end 75-bp sequencing at the Genomics Laboratory of The Walter and Eliza Hall Institute of Medical Research on a NextSeq 500 next-generation sequencer (Illumina) to obtain ∼20 million reads per sample.
6
Bacterial gDNA and RNA Extraction
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gDNA was isolated from bacteria grown on blood agar plates using the Genomic-tip 100/G kit (Qiagen, Hilden, Germany) following the manufacturer's protocol. The gDNA pellet was dissolved over night at room temperature with EB buffer.
For RNA extraction, 5 ml of bacterial cells grown in liquid medium were pelleted (4°C, 6000 × g, 3 min), snap-frozen in liquid nitrogen and stored at −80°C. Afterwards, bacterial pellets were disrupted with a FastPrep® FP120 Cell Disrupter (Thermo Savant) using Lysing Matrix B 2 ml tubes containing 0.1 mm silica beads (MP Biomedicals, Eschwege, Germany). Isolation of RNA was performed using the RNeasy kit (Qiagen, Hilden, Germany) and on-column DNase digestion with DNase I. A second DNase treatment was carried out using the TURBO DNA-free™ Kit (Ambion, Kaufungen, Germany). Isolated RNA was checked for the absence of DNA contamination by PCR.
DNA and RNA concentrations were measured using a NanoDrop 2000 spectrophotometer (Peqlab Biotechnologies). RNA quality given as RINe number was measured with an Agilent 4200 Tape Station system using RNA Screen Tapes (Agilent, Waldbronn, Germany). All RINe numbers of RNA preparations used for further processing were higher than 8.2, confirming high quality and little RNA degradation.
For RNA extraction, 5 ml of bacterial cells grown in liquid medium were pelleted (4°C, 6000 × g, 3 min), snap-frozen in liquid nitrogen and stored at −80°C. Afterwards, bacterial pellets were disrupted with a FastPrep® FP120 Cell Disrupter (Thermo Savant) using Lysing Matrix B 2 ml tubes containing 0.1 mm silica beads (MP Biomedicals, Eschwege, Germany). Isolation of RNA was performed using the RNeasy kit (Qiagen, Hilden, Germany) and on-column DNase digestion with DNase I. A second DNase treatment was carried out using the TURBO DNA-free™ Kit (Ambion, Kaufungen, Germany). Isolated RNA was checked for the absence of DNA contamination by PCR.
DNA and RNA concentrations were measured using a NanoDrop 2000 spectrophotometer (Peqlab Biotechnologies). RNA quality given as RINe number was measured with an Agilent 4200 Tape Station system using RNA Screen Tapes (Agilent, Waldbronn, Germany). All RINe numbers of RNA preparations used for further processing were higher than 8.2, confirming high quality and little RNA degradation.
7
Comprehensive RNA Isolation and Quality Control
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Total mRNA was isolated from both cells (HME1 and HME1) (AK + I) and then submitted to MedGenome Labs Pvt Ltd., Bangalore, India, by the local dealer: Genetrics, Dubai, UAE. RNA samples were quantified using Qubit RNA Assay HS (Invitrogen, Cat# Q32852). RNA purity was checked using QIAxpert, and RNA integrity was assessed on TapeStation using RNA ScreenTapes (Agilent Technologies, Santa Clara, CA, USA, Cat# 5067-5579). All RNA samples passed the QC and were passed for the RNA library prep.
8
RNA Extraction from Plucked Hairs and Skin Biopsies
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About 25–30 plucked hairs and about one third of each skin biopsy were taken out from the RNA-later buffer and used for RNA extraction. The samples were first homogenized using Precellys hard-tissue homogenizing tubes with ceramic beads and homogenized on a Precellys Evolution instrument (Bertin Instruments, Montigny-le-Bretonneux, France). After homogenization, the RNeasy kit (Qiagen, Hilden, Germany) was used to extract total RNA. DNase I was used during the process to digest genomic DNA from the total RNA samples. RNA purity and concentration were quantified on a NanoDrop instrument (Thermo Fisher Scientific). RNA quality was measured on a TapeStation instrument using the high sensitivity RNA screenTapes (Agilent, Santa Clara, CA, USA).
9
RNA-seq Analysis of C2C12 Cells
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RNA was isolated from C2C12 cells using a RNeasy Mini Kit, according to the manufacturer’s instructions. RNA integrity was measured using an Agilent 2200 TapeStation and RNA Screen Tapes (Agilent Technologies). Sequencing libraries were prepared using a NEBNext Ultra II RNA Library Kit for Illumina (New England Biolabs) with the NEBNext Poly (A) mRNA Magnetic Isolation Module (New England Biolabs), according to the manufacturer’s protocol. Prepared libraries were run on an Illumina HiSeq X sequencing platform in 150 bp paired-end mode. Sequencing reads were aligned to the GRCm38 mouse genome assembly using STAR (c.2.5.3). Mapped reads were counted for each gene using the GenomonExression pipeline (https://github.com/Genomon-Project/GenomonExpression ). Normalization of the read counts of RNA seq data and differential expression analysis were performed using the Bioconductor package DESeq2 (version 1.26.0). Differentially expressed genes with a greater than twofold change and a false discovery rate less than 0.1 were filtered and evaluated. RNA seq data have been deposited with links to BioProject accession number PRJDB17426 in the DDBJ BioProject database.
10
Gallbladder Cancer Tissue Profiling
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The study was approved by All India Institute of Medical Sciences, Rishikesh institutional ethical board (177/IEC/IM/2019). After obtaining informed written consent from the GBC patients and healthy controls, the cancer tissue (2 in number) was obtained from excised gallbladder during surgery and stored in RNA later at − 20 °C until the experiments were performed. All methods and procedures conducted during the course of this study were performed in accordance with applicable ethical standards, guidelines, and regulations. Gallbladder tissue excised as a part of specimen in surgery, e.g., Whipple’s procedure, right hepatic resection, etc., for normal non-dysplastic gallbladder (3 in number) was taken as a control. The miRNeasy Mini Kit (Qiagen, Cat#217004) was used to extract RNA from these specimens, and the Qubit RNA BR Assay (Invitrogen, Cat# Q10211) was used to quantify all samples. QIAxpert was used to examine RNA quality, and Tape Station was used to analyse RNA integrity using RNA screen Tapes (Agilent, Cat# 5067–5576).
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