We collected whole heart specimens from cKO and floxed-control adult animals at 22 weeks of age to include: three male cKO, three male control, three female cKO, and three female control samples. Gene microarray analysis was conducted as previously described (34 (link)). Total RNA was isolated using TRI Reagent (Molecular Research Center, Part # TR118). After isolation, RNA was quantified and treated with RNase free DNase I (Epicentre; Part # D9905K) and repurified using RNA Clean XP magnetic beats (Beckman Coulter Part # A63987). Amplified cDNA libraries were prepared from 200 ng of DNA-free total RNA using the Universal Plus mRNA-Seq Library Prep Kit (NuGEN Technologies, Inc; Part #0509-96). Chip-based capillary electrophoresis on Agilent 2100 Bioanalyzer High Sensitivity DNA assays (Agilent Technologies; Part # 5067-4626) was used to analyze quality of the libraries, which were then quantified with the Takara Library Quantification Kit (Part # 638324).
Universal plus mrna seq library prep kit
Manufactured by Tecan
Sourced in United States
The Universal Plus mRNA-Seq Library Prep Kit is a lab equipment product designed for the preparation of mRNA-Seq libraries. It provides the core functionality for performing mRNA-Seq library preparation.
Automatically generated - may contain errors
Lab products found in correlation
Agilent 2100 bioanalyzer high sensitivity dna assay, by Agilent Technologies (4 mentions)
Agilent 2100 bioanalyzer rna 6000 pico assay, by Agilent Technologies (3 mentions)
P3 flow cell, by Illumina (3 mentions)
P3 300 cycle reagent kit, by Illumina (3 mentions)
Mirneasy micro kit, by Qiagen (3 mentions)
Rnase free dnase 1, by Illumina (2 mentions)
Library quantification kit, by Agilent Technologies (1 mentions)
Tri reagent, by Molecular Research Center (1 mentions)
Hiseq 4000, by Illumina (1 mentions)
Mirneasy mini kit, by Qiagen (1 mentions)
Rneasy minelute column, by Qiagen (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Nextseq 2000 instrument, by Illumina (1 mentions)
6 protocols using universal plus mrna seq library prep kit
1
Gene Expression Analysis of Whole Heart Samples
2
Transcriptomic profiling of lung tissue
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Total lung RNA was isolated from frozen tissue with a miRNeasy mini kit (Qiagen). RNA samples of 100 ng were used to prepare amplified cDNA libraries using the Universal Plus mRNA-Seq Library Prep Kit (NuGEN Technologies, Inc.). Libraries were then sequenced with 2 × 150 bp paired-end configuration on Illumina HiSeq 4000 with a single index read. RNA-seq datasets have been deposited in SRA (PRJNA637249).
3
RNA-seq Analysis of NPC Transcriptome
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Total RNA was extracted from SVZ-derived NPCs using RNeasy Mini Kit (Qiagen), digested with RNAse-free DNAse I (Epicentre), and purified using RNeasy MinElute columns (Qiagen). cDNA libraries were constructed from DNA-free total RNA (50 ng) using the Universal Plus mRNA-Seq Library Prep Kit (NuGEN Technologies), followed by loading onto an Illumina NextSeq 500 v2.5 flow cell cartridge. The libraries were extended, and bridge amplified to create sequence clusters and sequenced with 76 nt paired-end reads plus 8 nt single-index reads using the Illumina NextSeq 500 High Output sequencing reagent kit v2 controlled by the NextSeq Control Software version 2.2.0.4. DESeq2 was employed to examine the effect of METH, EcoHIV, and the interaction of METH and EcoHIV on gene expression level [21 (link)]. Library preparation, sequencing, and generating FASTQ files were performed by Ocean Ridge Biosciences (http://www.oceanridgebio.com/ ) [22 (link)].
4
RNA-seq Analysis of Virus Infection
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For RNAseq analysis, three independent replicates were prepared for each treatment groups: Mock, MOI 0.01- and MOI 0.1-infected cultures, at 6 and 24 hpi. Total RNA was isolated using the miRNeasy micro kit (Qiagen) according to the manufacturer’s instructions. The RNA was quantified by O.D. measurement before being assessed for quality by chip-based capillary electrophoresis using Agilent 2100 Bioanalyzer RNA 6000 Pico assays (Agilent Technologies; Part # 5067-1513).
Libraries were prepared from 150 nanograms (ng) of DNA-free total RNA using the Universal Plus mRNA-Seq Library Prep Kit (NuGEN Technologies, Inc.; Part # 0508–96). The quality and size distribution of the amplified libraries were determined by chip-based capillary electrophoresis on Agilent 2100 Bioanalyzer High Sensitivity DNA assays (Agilent Technologies; Part # 5067–4626). Libraries were quantified using the Takara Library Quantification Kit (Part # 638324). The libraries were pooled at equimolar concentrations and diluted prior to loading onto a P3 flow-cell (Illumina; Part # 20027800) with the P3 300 Cycle reagent kit (Illumina; Part # 20038732) on the NextSeq2000 instrument.
Libraries were prepared from 150 nanograms (ng) of DNA-free total RNA using the Universal Plus mRNA-Seq Library Prep Kit (NuGEN Technologies, Inc.; Part # 0508–96). The quality and size distribution of the amplified libraries were determined by chip-based capillary electrophoresis on Agilent 2100 Bioanalyzer High Sensitivity DNA assays (Agilent Technologies; Part # 5067–4626). Libraries were quantified using the Takara Library Quantification Kit (Part # 638324). The libraries were pooled at equimolar concentrations and diluted prior to loading onto a P3 flow-cell (Illumina; Part # 20027800) with the P3 300 Cycle reagent kit (Illumina; Part # 20038732) on the NextSeq2000 instrument.
5
RNA-Seq Analysis of HBMEC Cultures
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For RNA-Seq analysis, three independent replicates were prepared for each treatment group, Mock, MOI 0.01-, and MOI 0.1-exposed HBMEC cultures, after 6 and 24 h. Total RNA was isolated via the miRNeasy micro kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The RNA was quantified by O.D. measurement before being assessed for quality by chip-based capillary electrophoresis using Agilent 2100 Bioanalyzer RNA 6000 Pico assays (Agilent Technologies, St Clara, CA, USA; Part # 5067-1513).
Libraries were prepared from 150 nanograms (ng) of DNA-free total RNA using the Universal Plus mRNA-Seq Library Prep Kit (NuGEN Technologies, Inc., San Francisco, CA, USA; Part # 0508-96). The quality and size distribution of the amplified libraries was determined by chip-based capillary electrophoresis on Agilent 2100 Bioanalyzer High Sensitivity DNA assays (Agilent Technologies; Part # 5067-4626). Libraries were quantified using the Takara Library Quantification Kit (Shiga, Japan; Part # 638324). The libraries were pooled at equimolar concentrations and diluted prior to loading onto a P3 flow cell (Illumina, San Diego, CA, USA; Part # 20027800) with the P3 300 Cycle reagent kit (Illumina, San Diego, CA; Part # 20038732) on the NextSeq2000 instrument (Illumina, San Diego, CA, USA).
Libraries were prepared from 150 nanograms (ng) of DNA-free total RNA using the Universal Plus mRNA-Seq Library Prep Kit (NuGEN Technologies, Inc., San Francisco, CA, USA; Part # 0508-96). The quality and size distribution of the amplified libraries was determined by chip-based capillary electrophoresis on Agilent 2100 Bioanalyzer High Sensitivity DNA assays (Agilent Technologies; Part # 5067-4626). Libraries were quantified using the Takara Library Quantification Kit (Shiga, Japan; Part # 638324). The libraries were pooled at equimolar concentrations and diluted prior to loading onto a P3 flow cell (Illumina, San Diego, CA, USA; Part # 20027800) with the P3 300 Cycle reagent kit (Illumina, San Diego, CA; Part # 20038732) on the NextSeq2000 instrument (Illumina, San Diego, CA, USA).
6
RNA-seq Library Preparation for Viral Infection
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For RNAseq analysis, three independent replicates were prepared for each treatment groups: Mock, MOI 0.01- and MOI 0.1-infected cultures, at 6 and 24 hpi. Total RNA was isolated using the miRNeasy micro kit (Qiagen) according to the manufacturer's instructions. The RNA was quantified by O.D. measurement before being assessed for quality by chip-based capillary electrophoresis using Agilent 2100 Bioanalyzer RNA 6000 Pico assays (Agilent Technologies; Part # 5067 – 1513).
Libraries were prepared from 150 nanograms (ng) of DNA-free total RNA using the Universal Plus mRNA-Seq Library Prep Kit (NuGEN Technologies, Inc.; Part # 0508 – 96). The quality and size distribution of the amplified libraries were determined by chip-based capillary electrophoresis on Agilent 2100 Bioanalyzer High Sensitivity DNA assays (Agilent Technologies; Part # 5067 – 4626). Libraries were quantified using the Takara Library Quantification Kit (Part # 638324). The libraries were pooled at equimolar concentrations and diluted prior to loading onto a P3 flow-cell (Illumina; Part # 20027800) with the P3 300 Cycle reagent kit (Illumina; Part # 20038732) on the NextSeq2000 instrument.
Libraries were prepared from 150 nanograms (ng) of DNA-free total RNA using the Universal Plus mRNA-Seq Library Prep Kit (NuGEN Technologies, Inc.; Part # 0508 – 96). The quality and size distribution of the amplified libraries were determined by chip-based capillary electrophoresis on Agilent 2100 Bioanalyzer High Sensitivity DNA assays (Agilent Technologies; Part # 5067 – 4626). Libraries were quantified using the Takara Library Quantification Kit (Part # 638324). The libraries were pooled at equimolar concentrations and diluted prior to loading onto a P3 flow-cell (Illumina; Part # 20027800) with the P3 300 Cycle reagent kit (Illumina; Part # 20038732) on the NextSeq2000 instrument.
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