RNA was extracted from FFPE blocks using the truXTRAC FFPE total NA Ultra kit (Covaris). RNA quality with DV200 was examined using the 4200 TapeStation (Agilent Technologies), and RNA concentration was determined using the Qubit Fluorometric Quantitation (Thermo Fisher). Ribosome RNA was depleted using the QIAseq FastSelect (Qiagen), and stranded cDNA library for RNAseq was prepared using the TruSeq Stranded LT mRNA kit (Illumina), following the manufacturer’s protocols. Pooled libraries were paired-end sequenced (100 bp × 2) on the NextSeq 2000 system (Illumina). Raw sequence reads were de-multiplexed by the on-instrument DRAGEN (v3.8.4). Sequence reads of each sample were pseudo-aligned to the human hg38 reference transcriptome, and the gene transcript abundance was quantified using the Kallisto (v0.48.0). Differential gene expression was achieved using biomaRt (v2.50.3), tximport (v1.22.0) and DESeq2 (v1.34.0) packages in the R Studio (Build 386 with R v4.1.1).
Nextseq 2000 system
Manufactured by Illumina
Sourced in United States
The NextSeq 2000 system is a high-throughput sequencing platform designed for a wide range of applications. It utilizes sequencing-by-synthesis technology to generate DNA sequence data. The system is capable of producing large volumes of sequencing data efficiently.
Automatically generated - may contain errors
Lab products found in correlation
Qubit fluorometer, by Thermo Fisher Scientific (4 mentions)
Kapa hifi dna polymerase, by Roche (3 mentions)
Nebnext ultra 2 directional rna library prep kit, by New England Biolabs (2 mentions)
Bioanalyzer 2100 system, by Agilent Technologies (2 mentions)
Magmax cell free dna isolation kit, by Thermo Fisher Scientific (2 mentions)
Quantifluor dsdna system, by Promega (2 mentions)
Single cell lysis solution, by Thermo Fisher Scientific (2 mentions)
Nebnext ultra 2 directional rna second strand synthesis, by New England Biolabs (2 mentions)
Nebnext ultra 2 rna first strand synthesis, by New England Biolabs (2 mentions)
Qiaseq fastselect rrna hmr kit, by Qiagen (2 mentions)
Anti pe microbeads, by Miltenyi Biotec (2 mentions)
Nebnext poly a mrna magnetic isolation module and ultra 2 directional rna library prep kit for, by Illumina (2 mentions)
Nextseq p3 sequencing reagent kit, by Illumina (2 mentions)
Tapestation 4200, by Agilent Technologies (2 mentions)
Qiaseq fastselect, by Qiagen (1 mentions)
Truseq stranded mrna lt kit, by Illumina (1 mentions)
Qubit fluorometric quantitation, by Thermo Fisher Scientific (1 mentions)
Nebnext single cell low input rna library prep kit for, by Illumina (1 mentions)
Rneasy micro kit, by Qiagen (1 mentions)
Qubit high sensitivity kit, by Thermo Fisher Scientific (1 mentions)
30 protocols using nextseq 2000 system
1
FFPE RNA Extraction and Transcriptomic Analysis
2
RNA Extraction and RNA-seq Analysis of Cell Cultures
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The RNA extraction was realized on cell cultures once they reached the stationary growth phase by using the NucleoSpin RNA kit (Mascherey-Nagel). The cell lysis step was adapted, i.e., by using a lysosomes concentration 10 times higher than the one recommended in the kit. The quantity and quality of RNA extracted was measure by Qubit. The RNA sequencing was then performed by Seqalis.
The RNA-seq libraries were sequenced on an Illumina NextSeq 2000 system using 2 × 100 bp paired-end sequencing. The raw sequencing reads were pre-processed using Cutadapt v4.2 to remove adapter sequences and low-quality reads. The pre-processed reads were then aligned to the reference genome using STAR v2.7.10b with default parameters. Gene expression levels were estimated using featureCounts v2.0.3. The count data were imported into R using the DESeq2 package. Differential gene expression analysis was performed using DESeq2 with a false discovery rate (FDR) of less than 0.05 and an absolute log2-fold change greater than 1 considered significant. The differentially expressed genes were annotated using the Gene Ontology (GO) database and pathway analysis was performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database.
The RNA-seq libraries were sequenced on an Illumina NextSeq 2000 system using 2 × 100 bp paired-end sequencing. The raw sequencing reads were pre-processed using Cutadapt v4.2 to remove adapter sequences and low-quality reads. The pre-processed reads were then aligned to the reference genome using STAR v2.7.10b with default parameters. Gene expression levels were estimated using featureCounts v2.0.3. The count data were imported into R using the DESeq2 package. Differential gene expression analysis was performed using DESeq2 with a false discovery rate (FDR) of less than 0.05 and an absolute log2-fold change greater than 1 considered significant. The differentially expressed genes were annotated using the Gene Ontology (GO) database and pathway analysis was performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database.
3
Transcriptome Analysis of Tissue Samples
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Thirty-seven tissue samples from 19 animals (Table 1 ) were used for the analysis of their transcriptomes via RNA-sequencing (RNA-seq). Total RNA was isolated using the RNeasy Micro Kit (Qiagen), followed by a DNase digestion step. Library preparation was carried out upon mRNA enrichment with the NEBNext Single Cell/Low Input RNA Library Prep Kit for Illumina (New England BioLabs). Single read sequencing took place on a NextSeq 2000 System (Illumina), using the corresponding NextSeq 2000 P3 v3 chemistry, with a read length of 72 base pairs. The integrity of the RNA and the quality of the library were assessed using a TapeStation 4200 (Agilent).
4
Profiling KRAS G12 Variants in cfDNA
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The analyses of KRAS G12 variants in cfDNA were performed via NGS using the NextSeq 2000 System (Illumina, CA, USA). Approximately 40 ng cfDNA per sample was used for library preparation using the Trusight Oncology 500 Kit (Illumina) according to the manufacturer’s instructions. The sequence data were processed and analyzed using the TruSight Oncology 500 Local App version 1.3 (Illumina). The average sequencing depth was approximately 1500×. Variant allele frequencies (VAFs) were calculated as altered variant reads/total reads.
Genomic DNA was extracted from 6 × 10 μm tissue sections using the GeneRead DNA FFPE Kit (Qiagen, Venlo, The Netherlands). DNA concentrations were measured using a Qubit high-sensitivity kit (Thermo Fisher Scientific). Subsequently, 40 ng of DNA was used as input for library preparation. Input DNA was sheared on a Covaris M220 Focused-ultrasonicator (Covaris, MA, USA) using a microTUBE-50 AFA Fiber Screw-Cap (Covaris) following the manufacturer’s instructions. DNA libraries were prepared using the hybrid capture-based TruSight Oncology 500 Library Preparation Kit (Illumina) following Illumina’s assay protocol. Libraries were sequenced using NexSeq550 (Illumina).
Genomic DNA was extracted from 6 × 10 μm tissue sections using the GeneRead DNA FFPE Kit (Qiagen, Venlo, The Netherlands). DNA concentrations were measured using a Qubit high-sensitivity kit (Thermo Fisher Scientific). Subsequently, 40 ng of DNA was used as input for library preparation. Input DNA was sheared on a Covaris M220 Focused-ultrasonicator (Covaris, MA, USA) using a microTUBE-50 AFA Fiber Screw-Cap (Covaris) following the manufacturer’s instructions. DNA libraries were prepared using the hybrid capture-based TruSight Oncology 500 Library Preparation Kit (Illumina) following Illumina’s assay protocol. Libraries were sequenced using NexSeq550 (Illumina).
5
RNA Extraction and Sequencing from Mouse Heart
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RNA was extracted from freshly harvested mouse heart tissue with RNAzol Reagent, following the manufacturer’s protocol (Molecular Research Center, Inc. Cincinnati, OH, USA). At least 3 mice from each experimental group were utilized for RNA extraction. Libraries were constructed following an established protocol [25 (link)]. All the libraries were analyzed and quantified with a bioanalyzer and sequenced on an Illumina NEXTSeq 2000 system using a fee-based service at the Weill Cornell Genomic Core Facility. All sequence data of the 13 libraries were deposited in the NCBI Sequence Read Archive (SRA) database under the accession number PRJNA916203.
6
RNA-Seq for Transcriptional Profiling
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RNA sequencing was performed by StarSEQ GmbH, Mainz, Germany. The quality of the extracted RNA was verified by the company with a 2100 Bioanalyzer system (Agilent Technologies, Santa Clara, CA, USA). After mRNA isolation and library preparation using a NEBNext© Ultra™ II Directional RNA Library Prep Kit (New England Biolabs, Ipswich, MA, USA), RNA sequencing of around 25 Mio PE reads (2 × 12.5 M reads, 2 × 150 nt) was performed with an Illumina NextSeq 2000™ system. Each treatment and control group included two replicates.
7
RNA Extraction and Sequencing from Organoids
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RNA from untreated and infected organoids was extracted with TRIzol reagent according to the manufacturer’s protocol, with minor modifications. Briefly, washed retina organoids were resuspended in 700 μL TRIzol reagent. The tissue was disrupted mechanically by vortexing for 30 s and pipetting 20 times. Chloroform 140 μL was added and samples were mixed vigorously for 15 s. Following centrifugation at 4°C, the aqueous phase was carefully separated and RNA was further precipitated by 350 μL 2-propanol. RNA pellets were washed twice with 75% ethanol and resuspended in diethyl pyrocarbonate (DEPC)-treated water for further library preparation.
Total RNA 600 ng was used for RNA isolation, fragmentation, and cDNA synthesis using the NEBNExt poly(A) mRNA magnetic isolation module (E7490). cDNA libraries were amplified and index labeled using the NEBNext Ultra TM II RNA Library Prep kit for Illumina (E7770, E7775). DNA library quality and concentration were analyzed on an Agilent Bioanalyzer DNA Chip. cDNA samples were loaded on a NextSeq 2000 system (Illumina) at a concentration of 800 pmol, and the sequencing was performed with single-end 100-bp reads.
Total RNA 600 ng was used for RNA isolation, fragmentation, and cDNA synthesis using the NEBNExt poly(A) mRNA magnetic isolation module (E7490). cDNA libraries were amplified and index labeled using the NEBNext Ultra TM II RNA Library Prep kit for Illumina (E7770, E7775). DNA library quality and concentration were analyzed on an Agilent Bioanalyzer DNA Chip. cDNA samples were loaded on a NextSeq 2000 system (Illumina) at a concentration of 800 pmol, and the sequencing was performed with single-end 100-bp reads.
8
Spatial Transcriptomics of Frozen Muscle Samples
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The fresh frozen samples in OCT media were sectioned to 10 μm using a cryostat and mounted on 10X Visium slides and stained with hematoxylin and eosin (H&E). The H&E stained samples underwent high-resolution imaging on the confocal Dragonfly Spinning Disk and Super Resolution Microscope at the Bioimaging center of Delaware Biotechnology Institute (DBI) and were subsequently visualized using Fiji software69 (link). After imaging, muscle samples were permeabilized and cDNA libraries were generated following the manufacturer’s instructions using Visium Spatial Gene Expression Slide & Reagent Kit (10x Genomics, PN-1000187; Pleasanton, CA). Quality analysis of the extracted total RNA and cDNA was conducted using the fragment analyzer to verify both quality and quantity, including Qubit quantification. The libraries were then sequenced in paired-end 150 bp mode on an Illumina Nextseq 2000 system (Illumina, San Diego,CA), with sequencing depth determined by the percentage capture areas covered by each muscle sample.
9
Total RNA-seq Library Preparation
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The cDNA library preparation and sequencing for the total RNA-seq analysis were carried out by the Core Unit Systems Medicine (Core Unit SysMed) of the Medical Faculty of the University of Würzburg and the Interdisciplinary Center for Clinical Research of the University Hospital Würzburg. First, ribosomal RNA was removed using RiboCOP META depletion kit followed by ultrasound treatment (one pulse of 30 s at 4°C). An adapter was then ligated to the 3′ end of the fragmented RNA molecules. For the synthesis of first-strand cDNA, the M-MLV reverse transcriptase was used; the introduced 3′-adapter served as a primer. The 5′ Illumina TruSeq sequencing adapters were ligated to the cDNA, and the cDNA was PCR amplified (10–20 ng µL−1). The amplified cDNA was purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics) and subsequently assessed via capillary electrophoresis. The cDNA was pooled and further purified through preparative agarose gel electrophoresis leading to fragments of cDNA ranging from 200 to 600 bp. The resulting pooled libraries were sequenced using an Illumina NextSeq 2000 system with 100 bp read length.
10
Transcriptome and miRNA Sequencing Analysis
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mRNA-seq and miRNA-seq were prepared using the TruSeq Stranded mRNA Sample Preparation kit (Illumina, San Diego, CA, USA) and QIAseq miRNA Library Kit (Qiagen) according to the manufacturer’s instructions, respectively. Optionally, size selection of library for miRNA sequencing was performed using 6% TBE polyacrylamide gel (Invitrogen). Electrophoresis was performed at 120 V for 1 h in Tris–borate-EDTA (TBE) buffer, and the band of interest (173 bp) was eluted using a Spin-X Centrifuge Tube Filter column (Corning Caster).
Each library was quantified by qPCR using the KAPA Library Quantification kit (KAPA). The quantified libraries were mixed at equimolar ratios, and the diluted library pool was loaded onto a NextSeq 1000/2000 P2 reagent v3 (100 Cycles) cartridge with a mixture of 75 pM and 1% phiX, and run on a NextSeq 2000 system (Illumina) in paired-end × 100 nucleotide multiplex and sequenced according to the manufacturer’s instructions. The resulting FASTQ files were analyzed with BaseSpace DRAGEN RNA Pipeline (Illumina) and Gene Globe (Qiagen).
Each library was quantified by qPCR using the KAPA Library Quantification kit (KAPA). The quantified libraries were mixed at equimolar ratios, and the diluted library pool was loaded onto a NextSeq 1000/2000 P2 reagent v3 (100 Cycles) cartridge with a mixture of 75 pM and 1% phiX, and run on a NextSeq 2000 system (Illumina) in paired-end × 100 nucleotide multiplex and sequenced according to the manufacturer’s instructions. The resulting FASTQ files were analyzed with BaseSpace DRAGEN RNA Pipeline (Illumina) and Gene Globe (Qiagen).
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