RNA-seq was performed as previously described [40 (link)], with few modifications. Briefly, the DNF-471 Standard Sensitivity RNA Analysis Kit, run on Fragment Analyzer (both from Advanced Analytical, Ankeny IA) was used to check RNA quality. Libraries were prepared from total RNA using TruSeq RNA Sample Preparation v2 according to the manufacturer's protocol (Illumina, San Diego, CA). Samples were sequenced on Illumina HiScanSQ platform. Sequencing reads were trimmed out of the low-quality bases with Fastx Toolkit (HannonLab, CHSL). Filtered sequences were mapped on hg19 genome assembly by using TopHat v2.0.6 and mRNA quantification was performed using Cuffdiff v2.0.2. For downstream analysis, genes with RPKM < 1 in all the samples were filtered out. Custom scripts on R software were used for clustering and heatmap analysis (https://www.r-project.org/ ). Gene Ontology was analyzed by using Database for Annotation, Visualization and Integrated Discovery (DAVID) program (https://david.ncifcrf.gov/ ).
Standard sensitivity rna analysis dnf 471 kit
Manufactured by Agilent Technologies
Sourced in Germany
The Standard Sensitivity RNA Analysis DNF-471 Kit is a laboratory equipment product from Agilent Technologies. It is designed for the analysis of RNA samples using standard sensitivity detection methods.
Automatically generated - may contain errors
Lab products found in correlation
Quant it broad range rna assay kit, by Thermo Fisher Scientific (4 mentions)
Rlt buffer, by Qiagen (3 mentions)
48 channel fragment analyzer, by Agilent Technologies (3 mentions)
Rneasy 96 kit, by Qiagen (2 mentions)
96 channel fragment analyzer, by Agilent Technologies (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Hiscansq platform, by Illumina (1 mentions)
Truseq rna sample preparation v2, by Illumina (1 mentions)
Fragment analyzer, by Agilent Technologies (1 mentions)
Paxgene blood rna extraction kit, by Qiagen (1 mentions)
Rna clean concentrator 5 kit, by Zymo Research (1 mentions)
Qubit 4 fluorometer, by Thermo Fisher Scientific (1 mentions)
Fragment analyzer automated ce system, by Agilent Technologies (1 mentions)
Qubit rna br assay kit, by Thermo Fisher Scientific (1 mentions)
Cytation 5 microplate reader, by Agilent Technologies (1 mentions)
12 channel fragment analyzer, by Agilent Technologies (1 mentions)
Cytation 5 instrument, by Agilent Technologies (1 mentions)
Allprep dna rna mini kit, by Qiagen (1 mentions)
β mercaptoethanol, by Qiagen (1 mentions)
Mirneasy micro kit, by Qiagen (1 mentions)
10 protocols using standard sensitivity rna analysis dnf 471 kit
1
RNA-seq Protocol for Transcriptome Analysis
2
RNA Extraction from PaxGene Blood Samples
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RNA extraction from blood collected in PaxGene tubes was performed via a column-based RNA extraction using the PaxGene blood RNA extraction kit (Qiagen). To optimize RNA concentrations, we used the RNA clean & concentrator-5 kit (Zymo research). We verified the RNA quality using a fragment analyzer (Advanced Analytical, DNF-471 Standard Sensitivity RNA Analysis Kit). No RNA samples had to be excluded based on low quality.
3
ZIKV RNA Fragmentation Analysis
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Viral RNA was fragmented by using 10× fragmentation buffer (NEB) and the recommended protocol. Briefly, the ZIKV RNA obtained from in vitro transcription (IVT) was mixed with fragmentation buffer (1× final) and then incubated at 94°C in a thermal cycler for 1, 3, 6, or 9 min. RNA fragmentation analyzer (Agilent, model 5003) was used to quantify the length distribution of RNA fragments by using the DNF-471 Standard Sensitivity RNA Analysis Kit (Fig. 2B ).
4
Isolating High-Quality Total RNA
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The same batch of vehicle- and AF-treated cells was collected and pelleted to isolate total RNA using the RNA Plus Mini Kit, according to the manufacturer's instructions. RNA concentration was checked using the Qubit RNA BR Assay Kit on Qubit 4 Fluorometer (ThermoFisher). The quality of the RNA samples was determined using the Fragment Analyzer Automated CE System (Agilent, DNF-471 Standard Sensitivity RNA Analysis Kit) with the High Sensitivity NGS Fragment Analysis Kit. No samples had to be excluded based on low quantity or quality.
5
RNA Extraction from HEK293 and HCT116 Cells
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HEK293 and HCT116 cells were seeded in a 12-well dish (2.5 million per well), 24 hours prior to RNA isolation. To extract RNA, media was removed from the culture dish and 200 μl of RLT buffer (Qiagen) was added directly to the cell layer. After complete lysis, lysates were transferred to microtubes and stored at -80°C until further processing. RNA was later purified from lysates using the RNeasy 96 Kit (Qiagen) according to the manufacturer's protocol. Residual DNA was removed by on-column DNase I treatment and RNA was eluted in 60 μl RNasefree water. Total RNA was quantitatively and qualitatively assessed using the fluorescence-based Broad Range Quant-iT RNA Assay Kit (Ther-moFisher) on a Cytation 5 microplate reader (BioTek) and the DNF-471 Standard Sensitivity RNA Analysis Kit on a 96-channel Fragment Analyzer (Agilent), respectively.
6
Isolating High-Quality MDSC RNA
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FACS-sorted CD11b+CD11c+ and CD11b+CD11c− MDSCs were collected in RLT buffer (QIAGEN, Hilden, Germany) supplemented with 2-mercaptoethanol followed by RNA extraction using the RNeasy Mini Kit (Qiagen) and adding an on-column DNA digestion step according to manufacturer’s instructions. Total RNA was quantitatively and qualitatively assessed using the absorbance-based Take3 microvolume plate system on a Cytation 5 instrument (BioTek, Bad Friedrichshall, Germany) and the Standard Sensitivity RNA Analysis DNF-471 Kit on a 12-channel Fragment Analyzer (Agilent Technologies, Santa Clara, CA, USA), respectively. Concentrations averaged at 310 ng/µl while RIN values ranged from 8.6 to 10, with an average of 9.8.
7
High-Quality RNA Extraction Protocol
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One million cells with viability of over 90% by Trypan blue measurement were collected, centrifuged, washed in PBS, and lysed in RLT buffer (79216, Qiagen) before storage at -80°. RNA was later extracted using the Qiagen RNeasy Mini Kit (74104, Qiagen, United States) according to manufacturer’s specifications. An on-column DNA digestion (DNASE10-1SET, Sigma) was performed; and the final elution volume was 30 μl. Total RNA was quantitatively and qualitatively assessed using the fluorescence-based Broad Range Quant-iT RNA Assay Kit (Q33140, Thermo Fisher) and the Standard Sensitivity RNA Analysis DNF-471 Kit (DNF-471-0500, Agilent) on a 48-channel Fragment Analyzer (Agilent), respectively. Concentrations averaged at 25 ng/μl, while RNA integrity number (RIN) ranged from 8.2 to 10, with a median of 9.9.
8
RNA-seq Analysis of HCT116 Cell Lines
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HCT116WT and HCT116CD276KO cells were cultured and maintained in different culture flasks for further propagation. Five biological replicates were used per group for a total of 10 samples. 1 × 106 cells per sample were collected, centrifuged, washed two times with PBS. After removal of supernatant completely, cell pellets were immediately frozen at − 80 °C until further use. Total RNA extraction for Next-Generation Sequencing from cell pellets lysed in RLT plus buffer were performed by using the AllPrep DNA/RNA Mini Kit (80224, Qiagen) according to the company’s protocol. An on-column DNA digestion was performed, and the final elution volume was 30 µL.
Total RNA was quantitatively and qualitatively assessed using the fluorescence-based RNA Broad Range Qubit Kit (Thermo Fisher) and the Standard Sensitivity RNA Analysis DNF-471 Kit on a 48-channel Fragment Analyzer (Agilent), respectively. Concentrations averaged at 30 ng/µL while RIN were all > 8.5.
Total RNA was quantitatively and qualitatively assessed using the fluorescence-based RNA Broad Range Qubit Kit (Thermo Fisher) and the Standard Sensitivity RNA Analysis DNF-471 Kit on a 48-channel Fragment Analyzer (Agilent), respectively. Concentrations averaged at 30 ng/µL while RIN were all > 8.5.
9
TGFβ1-Induced RNA Extraction Protocol
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After 72 h of TGFβ1 treatment in the co‐culture, cells were lysed in RLT buffer supplemented with 1% β‐mercaptoethanol (QIAGEN). Total RNA was extracted and purified on the MagMAX instrument (Thermo Fisher), using the RNeasy 96 Kit #74182 (QIAGEN) according to the manufacturer’s instructions. Total RNA was quantitatively and qualitatively assessed using the fluorescence‐based Broad Range Quant‐iT RNA Assay Kit (Thermo Fisher) and the Standard Sensitivity RNA Analysis DNF‐471 Kit on a 48‐channel Fragment Analyzer (Agilent), respectively. Concentrations averaged at 25.4 ng/µl while RIN ranged from 9 to 10, with a median of 10.
10
Transcriptomic Analysis of Rat Retinal Tissues
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A total of 28 days after treatment, rats were sacrificed with CO2. After enucleation, eyes were dissected on ice, neuroretinas and RPE/choroid complexes were collected in Eppendorf tubes, snap-frozen in nitrogen immediately, and then stored at −80 °C until the preparation for sequencing. Tissue samples were homogenized on dry ice in 200 µL Qiazol using a micro pestle for 10 s. After homogenization, 500 µL of Qiazol was added, and RNA isolation was performed with miRNeasy Micro Kit (Qiagen) according to manufacturer’s instructions, including an on-column DNase digestion. The quantity and quality of the 24 total RNA samples (12 RPE/Choroid, 12 neuroretinas) was assessed using the fluorescence-based Broad Range Quant-iT RNA Assay Kit (Thermo Fisher) and the Standard Sensitivity RNA Analysis DNF-471 Kit on a 96-channel Fragment Analyzer (Agilent), respectively. While the concentrations averaged at 83.7 ng/µL, the RIN ranged from 6.2 to 9.4.
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