DNA was extracted using the Qiagen AllPrep DNA/RNA Mini Kit. Sequencing of exome samples was performed using Illumina’s next-generation sequencing methodology [32 (link)]. In detail, total DNA was quality checked using Agilent 4200 TapeStation System (Agilent Genomic DNA ScreenTape) and quantified using Quant-iT™ PicoGreen™. Libraries were prepared from 3 µg of input material using SureSelect Human All Exon V6 (manufacturer’s instructions) and subsequently quantified and quality checked using Agilent 4200 TapeStation System (D1000 ScreenTape). Libraries were pooled and sequenced on NextSeq 500 System (High Output Flow Cell) running in 150 cycle (2 × 75 bp paired-end) mode. Sequence information was converted to FASTQ format using bcl2fastq v2.20.0.422. The WES data were used to search for HLA-presented individual neoepitopes in the complete OPSCC HLA ligandome dataset of each patient with available whole exome sequencing data (38/40 patients). Database search and spectral annotation were performed against the combination of the human proteome as comprised in the UniProtKB/Swiss-Prot database and the mutated protein sequences as defined for the respective patients.
Agilent 4200 tapestation system
Manufactured by Agilent Technologies
Sourced in United States, Germany, Canada, Netherlands
The Agilent 4200 TapeStation System is a compact and automated electrophoresis instrument designed for the analysis of nucleic acid samples. It provides a fast and reliable method for the assessment of DNA, RNA, and genomic DNA quality and size.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (15 mentions)
Nextseq 500, by Illumina (14 mentions)
Novaseq 6000, by Illumina (13 mentions)
Trizol reagent, by Thermo Fisher Scientific (10 mentions)
Trizol, by Thermo Fisher Scientific (8 mentions)
Ampure xp bead, by Beckman Coulter (8 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (7 mentions)
Turbo dna free kit, by Thermo Fisher Scientific (7 mentions)
Qubit 4.0 fluorometer, by Thermo Fisher Scientific (6 mentions)
Hiseq 4000, by Illumina (6 mentions)
Qubit 3, by Thermo Fisher Scientific (6 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (5 mentions)
Nextseq 500 550 high output kit v2, by Illumina (5 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (5 mentions)
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (5 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (5 mentions)
Qiaamp dna mini kit, by Qiagen (4 mentions)
Rneasy micro kit, by Qiagen (4 mentions)
Nextseq 500 platform, by Illumina (4 mentions)
Chromium single cell controller, by 10x Genomics (4 mentions)
188 protocols using agilent 4200 tapestation system
1
Exome Sequencing and Neoepitope Analysis
2
Isolation and Quantification of Circulating Cell-Free DNA
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Genomic DNA was extracted from tumor tissue using the Qiagen Puregene Core kit A (Qiagen) or the QIAamp DNA Mini kit (Qiagen) according to the manufacturer’s instructions and quantified on a Qubit 2.0 fluorometer (Life Technologies). Fragmentation of genomic DNA was achieved by 5 U of AluI or HaeIII restriction enzyme (New England Biolabs) added to each droplet digital PCR (ddPCR) reaction [14 (link)]. Thawed blood and bone marrow plasma, CSF, and urine samples were centrifuged at 2000× g for 5 min to clear debris, then supernatants were centrifuged at 20,000× g for 5 min. Cell-free DNA was purified from a minimum of 40 µL stored samples using the QIAamp Circulating Nucleic Acid kit (Qiagen), then concentrated to 50 μL using the DNA Clean and Concentrator-5 kit (Zymo Research), both according to manufacturers’ directions. Total cfDNA was quantified using the cfDNA ScreenTape assay (Agilent) and Agilent 4200 TapeStation System according to the manufacturer’s instructions [17 (link)]. DNA size distribution was likewise assessed with the Agilent 4200 TapeStation System [17 (link)]. DNA fragments between 100 and 300 bp were considered to be total cfDNA [39 (link)]. Assessment of DNA size distribution and cfDNA quantity does not allow any conclusion to be drawn about the cell(s) of origin.
3
RNA Extraction and Sequencing Protocol
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Total RNA was isolated using Qiagen RNeasy Micro kit and following the instruction. The integrity of the isolated RNA was examined by the Agilent 4200 TapeStation System. Libraries for RNA-Seq were constructed with KAPA RNA HyperPerp Kit to generate strand-specific RNA-seq libraries. The workflow consists of ribosome RNA (rRNA) depletion, RNA fragmentation and double-stranded cDNA generation using a mixture of random priming, followed by end repair to generate blunt ends, adaptor ligation, strand selection, and PCR amplification to produce the final libraries. Amplified libraries were quantified by Qubit dsDNA HS (High Sensitivity) Assay Kit, and quality-checked by the Agilent 4200 TapeStation System. Different index adaptors were used for multiplexing samples in one sequencing lane. Sequencing was performed with Illumina HiSeq 3000 sequencer to produce 50 base-pair single-end reads (1 × 50 bp).
4
Smart-seq2 RNA-Seq Library Preparation
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Total RNA was purified using the ReliaPrep RNA Cell Miniprep System and RNA-Seq libraries were generated using the Smart-seq2 method (Picelli et al., 2014 (link)). Five ng of RNA were retrotranscribed, cDNA was PCR-amplified (15 cycles) and purified with AMPure XP beads. After purification, the concentration was determined using Qubit 3.0 and size distribution was assessed using Agilent 4200 TapeStation system. Then, the tagmentation reaction was performed starting from 0.5 ng of cDNA for 30 minutes at 55°C and the enrichment PCR was carried out using 12 cycles. Libraries were then purified with AMPure XP beads, quantified using Qubit 3.0, assessed for fragment size distribution on an Agilent 4200 TapeStation system. Sequencing was performed on an Illumina NextSeq500 or NovaSeq6000 (single-end, 75bp read length) following manufacturer’s instruction.
5
Genomic DNA Extraction from Blood
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Genomic DNA was extracted from whole blood using the QiaAmp DNA Mini Kit (Qiagen, Milan, Italy) according to the manufacturer’s protocol or with the salting-out method. The DNA concentration and purity were determined, respectively, by a Qubit 4.0 fluorometer (Thermo Fisher, Milan, Italy) and Nanodrop spectrophotometer (Thermo Fisher, Milan, Italy). DNA integrity was evaluated using the Agilent Genomic ScreenTape assay (Agilent Technologies, Milan, Italy) on the Agilent TapeStation 4200 system (Agilent Technologies, Milan, Italy).
6
Differential Gene Expression in FFPE Tumor and Myometrial Tissue
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RNA‐seq was performed by Genewiz (South Plainfield). Total RNA was extracted from FFPE tumor tissue (n = 3) and myometrial tissue (n = 3) using the QIAGEN RNeasy FFPE kit (Qiagen). RNA was quantified using a Qubit 2.0 fluorometer and RNA integrity was checked using the Agilent TapeStation 4200 system (Agilent Technologies). RNA sequencing libraries were prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina and following the manufacturer's instructions (NEB). Briefly, samples underwent ribosomal RNA depletion, stranded RNA library preparation, and multiplexing and cluster generation, followed by the Illumina HiSeq (Illumina) system at 2 × 150 base pairs for paired‐end sequencing in a high output mode. The clean reads were mapped to the Homo sapiens GRCh37 reference genome available on ENSEMBL using STAR aligner v.2.5.2b. Feature “Counts” within the Subread package v.1.5.2 was applied to calculate unique gene hit counts. After extraction of gene hit counts, the gene hit counts table was used for downstream differential expression analysis. DESeq2 was used to identify differential expression analysis between groups. Differentially expressed genes were identified using a threshold adjusted P‐value < .05 and absolute log2 fold change > 1. RNA sequence data were submitted to the NCBI Sequence Read Archive (SRA accession number: PRJNA649753).
7
Whole-Exome Sequencing of COVID-19 Patients
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Genomic DNA of COVID-19 patients was extracted from peripheral blood using a Maxwell® RSC Blood DNA Kit (Promega, Madison, WI, USA), and DNA concentration and purity were evaluated using a NanoDrop™ 8000 spectrophotometer. For library preparation, we measured the DNA concentration with a Qubit® DNA Assay Kit with a Qubit® 2.0 fluorometer (Life Technologies by Thermo Fisher Scientific, Waltham, MA, USA), and 1.0 μg of DNA was used for library preparation. Genomic DNA was sonicated to generate 180–250-bp fragments and then end-polished, A-tailed, and ligated with the full-length adapter with further PCR amplification. PCR products were purified using the AMPure XP system (Beckman Coulter, Brea, CA, USA) and quantified using the Agilent high-sensitivity DNA assay from the Agilent TapeStation 4200 system (Agilent Technologies, Santa Clara, CA, USA). The whole exome was captured with Agilent SureSelect Human All Exome V6 (Agilent Technologies, Santa Clara, CA, USA), and the sequencing was performed on an Illumina NovaSeq 6000 System (Illumina, San Diego, CA, USA).
8
Metagenomic DNA Extraction and Sequencing
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Total DNA extraction was performed using the Pow-erSoil® DNA Isolation Kit (Mobio Labs, Inc., Solana Beach, CA, USA) according to the manufacturer's guideline at SENAI Institute of Innovation in Biosynthetic and Fibers (SENAI CETIQT, Rio de Janeiro, RJ, Brazil). The quantitative and qualitative DNA analysis and metagenomic library preparation and sequencing were performed at Computational Genomics Unity Darcy Fontoura de Almeida (UGCDFA) of the National Laboratory of Scientific Computation (LNCC) (Petrópolis, RJ, Brazil). The DNA concentration and integrity were determined using Agilent TapeStation 4200 System (Agilent Technologies, USA) with Genomic DNA ScreenTape according to the manufacturer's instructions. Libraries were constructed using the Nextera DNA Flex library preparation kit (Illumina, USA) according to the manufacturer's recommendations. Sequencing was conducted on an Illumina NextSeq 500 platform (Illumina, San Diego, CA, USA) using the NextSeq 500/550 high-output kit v2.5 (Illumina, USA), with the system set to produce 2 × 150-bp reads.
9
RNA Extraction from Mouse Tissues
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Mice were sacrificed using cervical dislocation. Brain areas, whole brain (without olfactory bulb and brainstem) and liver tissue were dissected. Total RNA was prepared using 1 ml TRI Reagent® (SIGMA, T9424-200ml) for 100 mg tissue, followed by addition of 200 μl chloroform (Honeywell Riedel-de Haën, 34854-2.5L). After incubation (10 min) at room temperature (RT), tissue suspension was centrifuged (16 000 × g, 4°C, 15 min). The upper aqueous phase was taken and mixed in a 1:1 ratio with isopropanol (Honeywell Riedel-de Haën, 34965-4 × 2.5 l) and 1 μl glycogen (Thermo Fisher Scientific, R0551) was added, incubated at RT (5 min) and centrifuged (16 000 × g, 4°C, 5 min). The RNA pellet was washed twice with ice-cold 75% ethanol including centrifugation (16 000 × g, 4°C, 5 min). RNA was reconstituted in 50 μl nuclease-free water (Zymo Research, W1001-10). RNA concentrations were determined using UV-VIS spectrophotometer Nanodrop 2000 (Thermo Fisher Scientific, Waltham, USA) and the integrity of the RNA was checked using the Agilent TapeStation 4200 system (Agilent, Santa Clara, USA) analysis to obtain eRIN values.
10
Genomic DNA and RNA Extraction from Horsegram
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Genomic DNA (gDNA) was isolated from horsegram variety PHG-9 (Supplementary Figure S1 ) using the DNAeasy Plant Mini Kit as per the manufacturer’s instructions (Catalog # 69104, Qiagen), and the DNA quality was checked using a NanoDrop device. The RNA from root and leaf tissues was isolated using TRIzol reagent (Catalog # 15596026, Invitrogen), followed by the procedure of the Direct-zol RNA MiniPrep kit (Catalog # R2050, Zymo Research). The integrity and quantity of the RNA were checked using the Agilent 4200 TapeStation system. Then, the RNA from root and leaf tissues was mixed in equimolar proportions and processed as a single sample for RNA-seq library preparation.
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