Lung macrophages from PBS- and GEM-treated tumor-free mice were sorted, and RNA was extracted using a QIAGEN RNeasy Kit. The quantity of the purified RNA samples was measured by the RNA High Sensitivity Kit in the Qubit fluorometric quantification system (Thermo Fisher Scientific). Libraries were prepared using the Universal Plus mRNA-Seq with NuQuant (NuGEN). The pooled library was run on MiSeq to test quantity and quality using the MiSeq Reagent Nano Kit V2 300 cycles (Illumina). Sequencing was performed on the Illumina NextSeq 500 using the NextSeq 500/550 75 cycle High Output Kit v2.5. Differential expression was performed using 2 tools, DESeq2 and Cuffdiff2. DEGs at P value cutoff 0.01, or q value cutoff of 0.01 with log2 fold-change of 0 for the pairwise comparisons, were used for further analysis of enriched GO biological processes. A volcano plot was created to examine the distribution of log2 fold-change at different significance levels. RNA-Seq data were deposited with National Center for Biotechnology Information Gene Expression Omnibus accession GSE217105.
Nuquant
Manufactured by Tecan
NuQuant is a quantification solution for nucleic acid samples. It provides accurate and reliable measurements of DNA and RNA concentrations.
Automatically generated - may contain errors
Lab products found in correlation
Universal plus mrna seq, by Tecan (5 mentions)
Novaseq 6000 s4 flow cell, by Illumina (3 mentions)
Fragment analyzer, by Agilent Technologies (3 mentions)
Fastselect, by Qiagen (3 mentions)
Kingfisher flex system, by Thermo Fisher Scientific (3 mentions)
Quick rna magbead kit, by Zymo Research (3 mentions)
Nextseq 500, by Illumina (2 mentions)
Rneasy kit, by Qiagen (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Nextseq 500 550 high output kit v2 75 cycle, by Illumina (1 mentions)
Qubit fluorometric quantification system, by Thermo Fisher Scientific (1 mentions)
Rna high sensitivity kit, by Thermo Fisher Scientific (1 mentions)
Miseq reagent nano kit v2 300 cycles, by Illumina (1 mentions)
Facsaria 3, by BD (1 mentions)
Nextseq 2000 sequencer, by Illumina (1 mentions)
Kapa library quantification kit, by Roche (1 mentions)
Agilent 4200 tapestation, by Agilent Technologies (1 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (1 mentions)
Novaseq 6000 platform, by Illumina (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
9 protocols using nuquant
1
RNA-Seq Analysis of Lung Macrophages
2
Transcriptome Analysis of COVID-19 Neutrophils
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PBMCs from severe COVID-19 patients were washed and stained with viability dye–APC-Cy7, CD45-PerCP, CD66b-PE, CD16-APC for 30 minutes at 4°C. CD16hi and CD16Int CD66b+ neutrophils were sorted by a BD Biosciences FACSAria III. Cells were then lysed in TRIzol (Thermo Fisher Scientific), and RNAs were extracted with a QIAGEN RNeasy kit. Libraries were prepared using the Universal Plus mRNA-Seq with NuQuant (NuGen). Sequencing was performed on the University of Louisville Brown Cancer Center Genomics Core Illumina NextSeq 500 using the NextSeq 500/550 75 cycle high output kit v2.5. The RNA-Seq data have been deposited into NCBI’s Gene Expression Omnibus database with the accession number GSE154311.
3
Transcriptome Analysis of Cell Lines
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RNA extracted from cell lines was quantitated with the Qubit Fluorometer (Thermo Fisher Scientific) and screened for quality on the Agilent TapeStation 4200 (Agilent Technologies). The samples were then processed for RNA sequencing using the NuGEN Universal RNA-Seq Library Preparation Kit with NuQuant (NuGEN Technologies). Briefly, 50 ng of RNA was used to generate cDNA and a strand-specific library following the manufacturer's protocol. Quality control steps were performed, including TapeStation size assessment and quantification using the Kapa Library Quantification Kit (Roche). The final libraries were normalized, denatured, and sequenced on the Illumina NextSeq 2000 sequencer with the P3-200 cycle reagent kit to generate approximately 80–108 million base read pairs per sample (Illumina, Inc.).
4
Bulk RNA-seq for Donor-Cell Demultiplexing
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Bulk RNA-seq data was generated to extract genotype information, so that single-cells could be demultiplexed and matched to single donors. For each donor, RNA was extracted from PBMCs using the Quick RNA MagBead kit (Zymo Research) on a KingFisher Flex system (Thermofisher Scientific) according to the company’s protocol. RNA integrity was measured with the Fragment Analyzer (Agilent) and library generation was continued when integrity was at least 6. Total RNA-sequencing libraries were depleted from ribosomal and hemoglobin RNAs, and generated using FastSelect (Qiagen) and Universal Plus mRNA-seq with Nu Quant (Tecan) reagents. Pooled libraries were PE100 sequenced on an HiSeq4000 or PE150 sequenced on an Illumina NovaSeq 6000 S4 flow cell at the CZ Biohub.
5
Transcriptional Response to Interferon-α in LATS1 Knockdown
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Human fibroblast 2fTGH cells were transfected with either control short hairpin RNAs (shRNAs; shCON) or shRNAs against LATS1 (shLATS1). Seventy-two hours after transfection, cells were treated with IFN-α (1000 IU/ml) for 8 hours. Total RNAs were isolated from cells using a TRIzol reagent (Invitrogen). The RNA samples were sent to the Suzhou Institute of Systems Medicine in China for further sequencing analysis. The cDNA libraries were made for sequencing with Universal Plus mRNA-Seq with NuQuant (TECAN GENOMICS, 0520B-A01). Then, the libraries were sequenced on the NovaSeq 6000 platform (Illumina). The pair-end sequencing data were treated by fastp software to filter reads with bad quality and then mapped to Ensembl hg19 reference genome by CLC genomic workbench 12.0 (Qiagen). Differential expressed genes were defined as the genes whose fold changes (increase and decrease) are more than 2, with a P value <0.05 between two groups. The R package cluster Profiler and org.Hs.eg .db are the tool for Gene Ontology enrichment. All RNA-seq data have been deposited in the Gene Expression Omnibus (GSE193088).
6
High-Throughput DNA Sequencing Library Preparation
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DNA was extracted from approximately 5 × 106 cells
per sample using a QIAamp DNA Mini kit (Qiagen, Hilden, Germany) according
to the manufacturer’s protocol. DNA sequencing libraries were
prepared with a Tecan Celero EZ DNA-Seq library preparation kit (Männedorf,
Switzerland), starting from 100 ng of DNA, and with 20 min of enzymatic
fragmentation and seven PCR amplification cycles. Libraries were quantified
using NuQuant (Tecan) and normalized to 5 nM. The libraries were pooled
and sequenced on Illumina NovaSeq6000 paired-end flow cells (Illumina,
San Diego, CA) with 300 cycles using sequencing reagent kits.
per sample using a QIAamp DNA Mini kit (Qiagen, Hilden, Germany) according
to the manufacturer’s protocol. DNA sequencing libraries were
prepared with a Tecan Celero EZ DNA-Seq library preparation kit (Männedorf,
Switzerland), starting from 100 ng of DNA, and with 20 min of enzymatic
fragmentation and seven PCR amplification cycles. Libraries were quantified
using NuQuant (Tecan) and normalized to 5 nM. The libraries were pooled
and sequenced on Illumina NovaSeq6000 paired-end flow cells (Illumina,
San Diego, CA) with 300 cycles using sequencing reagent kits.
7
Bulk RNA-seq for Donor Demultiplexing
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Bulk RNA-seq data were generated to extract genotype information so that single cells could be demultiplexed and matched to single donors. For each donor, RNA was extracted from PBMCs using the Quick-RNA MagBead Kit (Zymo Research) on a KingFisher Flex system (Thermo Fisher Scientific) according to the manufacturer’s protocol. RNA integrity was measured with the Fragment Analyzer (Agilent), and library generation was continued when integrity was at least 6. Total RNA-seq libraries were depleted from ribosomal and hemoglobin RNAs and generated using FastSelect (Qiagen) and Universal Plus mRNA-Seq with NuQuant (Tecan) reagents. Pooled libraries were PE100-sequenced on a HiSeq 4000 or PE150-sequenced on an Illumina NovaSeq 6000 S4 flow cell at the Chan Zuckerberg Biohub.
8
Bulk RNA-seq for Single-cell Demultiplexing
9
RNA-Seq Library Preparation and Sequencing
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Cells were pre-treated with 0.1% DMSO or 100 nM cortistatin A for 30 minutes, then treated with 40 mM Tris, pH 7.4 or 10 ng/mL interferon gamma (Gibco, PHC4031). After four hours, total RNA was isolated from D21 or T21 cells using TRizol (Invitrogen, 15596026) as specified by the manufacturer and quantified with a Qubit® 3.0 using the RNA High Sensitivity (HS) kit (Invitrogen™, Q32855). 1 μg of total RNA with a RIN number of ≥8 was used for RNA-seq library prep. Libraries were constructed using Universal Plus™ mRNA-Seq library preparation kit with NuQuant® (Tecan, 0520). Library construction and sequencing were performed at the Genomics Shared Resource (CU Anschutz). Paired-end libraries (151 bp x 151 bp) were sequenced on the Illumina NextSeq 6000 platform (Firmware version 1.26.1, RTA version: v3.4.4, Instrument ID: A00405).
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