The samples were quantified using an Agilent 4200 Tapestation instrument, with a corresponding Agilent High Sensitivity RNA assay. The resulting RIN (RNA Integrity Number) scores and concentrations were taken into account for qualifying samples to proceed. The samples were then normalized to 2 ng/μL, before using KAPA’s mRNA HyperPrep library preparation kit. A final cleanup was performed using Aline PCRCLean DX beads in a 0.63x SPRI-based cleanup. The resulting purified libraries were run on an Agilent 4200 Tapestation instrument, with a corresponding Agilent High Sensitivity D1000 ScreenTape assay to visualize the libraries and to check the size and concentration of the library. Values obtained from this assay were used to normalize all samples in equimolar ratio. The pool was denatured and loaded onto an Illumina NextSeq instrument, with an Illumina NextSeq High Output 150-cycle kit to obtain Paired-End 75bp reads. The pool was loaded at 1.9 pM, with 5% PhiX spiked in to serve as a sequencing control. The basecall files were demultiplexed through the BioPolymer Facility’s pipeline, and the resulting FASTQ files were used in subsequent analysis.
Tapestation 4200
Manufactured by Agilent Technologies
Sourced in United States, Japan, United Kingdom, Germany
The TapeStation 4200 is a fully automated instrument that measures the size and quantification of nucleic acid samples. It uses microfluidic technology to analyze DNA and RNA samples in a rapid, sensitive, and reproducible manner. The TapeStation 4200 provides reliable sample quality information to support various downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Novaseq 6000, by Illumina (59 mentions)
Rneasy mini kit, by Qiagen (49 mentions)
Nextseq 500, by Illumina (47 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (37 mentions)
Hiseq 4000, by Illumina (25 mentions)
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (25 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (21 mentions)
2100 bioanalyzer, by Agilent Technologies (20 mentions)
Trizol, by Thermo Fisher Scientific (18 mentions)
Ampure xp bead, by Beckman Coulter (17 mentions)
Trizol reagent, by Thermo Fisher Scientific (17 mentions)
High sensitivity rna screentape, by Agilent Technologies (17 mentions)
Kapa library quantification kit, by Roche (16 mentions)
D1000 screentape, by Agilent Technologies (14 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (13 mentions)
Nanodrop, by Thermo Fisher Scientific (13 mentions)
Hiseq 2500, by Illumina (11 mentions)
Rneasy plus mini kit, by Qiagen (11 mentions)
Nextseq 550, by Illumina (11 mentions)
Miseq system, by Illumina (11 mentions)
625 protocols using tapestation 4200
1
Illumina NextSeq RNA-seq Library Prep
2
Comprehensive RNA-seq workflow for CT26 tumors
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CT26 tumor tissues were isolated and total RNA was extracted using the RNeasy 96 QIAcube HT Kit (Qiagen; C/N 74171) according to manufactuers guidelines with a DNase digest included. RNA concentration was determined by Qubit Flex Fluorometer (Invitrogen), RNA purity was determined using a NanoDrop Eight (Thermo Scientific) and RNA integrity was measured using a 4200 Tapestation (Agilent). All samples had a RINe of ≥7.0. Libraries were prepared using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England BioLabs; #E7760L) as per manufactuer’s guidelines with 1000 ng of RNA input. Ribosomal RNA was removed using the NEBNext® Poly(A) mRNA Magnetic Isolation Module (New England BioLabs; E7490L). Libraries were subjected to 9 cycles of PCR with unique dual indexes (New England BioLabs; E6440L). Libraries were subsequently quantified by Qubit Flex Fluorometer (Invitrogen), and library sizes were determined by 4200 Tapestation (Agilent) and pooled equimolar. Each library was loaded onto one lane of an S4 v1.5 flow cell (300 cycles) (Illumina, #20028312) on an Illumina NovaSeq 6000 at 2 × 150 paired-end configuration.
3
Transcriptome Profiling of Parasite-Infected Trout
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Total RNA was extracted from FACS-sorted T. bryosalmonae from brown trout and rainbow trout using RNeasy UCP Micro Kit (Qiagen) along with an on-column DNase digestion step following the manufacturer’s protocol. RNA integrity was assessed on the 4200 TapeStation with the High Sensitivity RNA ScreenTape Kit (Agilent, Santa Clara, CA, USA). Only samples with RNA integrity numbers > 8.0 were used for further analysis.
Library preparation was done with 60 ng total RNA input using the Poly(A) RNA Selection Kit V1.5 and the CORALL mRNA-Seq Library Prep Kit (Lexogen, Vienna, Austria) according to the manufacturers protocol. Six cDNA libraries (3 each for sorted parasite samples from brown trout and rainbow trout) were prepared. Library quality control was done with the High Sensitivity D1000 ScreenTape Kit on the 4200 TapeStation (Agilent). Libraries were sequenced on a NovaSeq 6000 system (Illumina, San Diego, CA, USA) implementing 150-bp paired-end reads. Sequencing was done by the NGS unit of the Vienna Biocenter Core Facilities (VBCF, Vienna, Austria).
Library preparation was done with 60 ng total RNA input using the Poly(A) RNA Selection Kit V1.5 and the CORALL mRNA-Seq Library Prep Kit (Lexogen, Vienna, Austria) according to the manufacturers protocol. Six cDNA libraries (3 each for sorted parasite samples from brown trout and rainbow trout) were prepared. Library quality control was done with the High Sensitivity D1000 ScreenTape Kit on the 4200 TapeStation (Agilent). Libraries were sequenced on a NovaSeq 6000 system (Illumina, San Diego, CA, USA) implementing 150-bp paired-end reads. Sequencing was done by the NGS unit of the Vienna Biocenter Core Facilities (VBCF, Vienna, Austria).
4
Genomic DNA extraction and sequencing
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Extracted genomic DNA (gDNA) was quantified by the Qubit 2.0 fluorometer (Thermo Fisher), and integrity was verified using a 4200 TapeStation (Agilent) genomic DNA assay. Sequencing libraries were prepared using the Qiaseq FX DNA library kit (Qiagen) per standard protocol. One microgram of gDNA was fragmented, Illumina adapter‐ligated, and amplified. The size selection for libraries was performed using SPRIselect beads (Beckman Coulter), and the purity of the libraries was analyzed using the high sensitivity DNA 1000 tape on the Tapestation 4200 (Agilent). Libraries were sequenced on a MiSeq (Illumina) and assembled using the SPAdes genome assembly algorithm.
5
Comprehensive Genomic DNA Extraction and Sequencing
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DNA was extracted with the AllPrep DNA/RNA Micro Kit (Qiagen, Hilden, Germany) and eluted in Tris–ethylenediaminetetraacetic acid buffer. The DNA concentration was determined by a Qubit (Invitrogen), the purity was determined by a NanoDrop 8000 (Thermo Fisher Scientific, Waltham, MA, USA), and DNA integrity was measured with a 4200 TapeStation (Agilent, Santa Clara, CA, USA). All samples had a DNA integrity number of ≥7.0. Libraries were prepared by using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA) and the SureSelect Human All Exon V8 (Agilent). Libraries were subsequently quantified by using the Qubit and KAPA library quantification kit, ROX low (Roche). Library sizes were also determined by a TapeStation 4200 (Agilent). Paired-end sequencing was performed on a NovaSeq 6000 (Illumina, San Diego, CA, USA). Reads were aligned to hg38 using bwa-0.7.17 with >60 million raw reads per sample.
6
RNA Sequencing Library Preparation
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RNA was quantified using the Quant-iT RiboGreen RNA Assay Kit (Thermo Fisher Scientific, R11490) and quality-checked with the RNA 6000 Nano Kit (Agilent, 5067–1511) on a 2100 Bioanalyzer (Agilent, G2939BA) or High Sensitivity RNA ScreenTape Assay (Agilent, 5067–5579, 5067–5580, 5067–5581) on a 4200 TapeStation (Agilent, G2991AA) prior to library generation. Libraries were prepared from total RNA with the TruSeq Stranded Total RNA Library Prep Gold Kit (Illumina, 20020599) according to the manufacturer’s instructions. Libraries were analyzed for insert size distribution with the High Sensitivity DNA Kit (Agilent, 5067–4626) on a 2100 Bioanalyzer or the High Sensitivity D1000 ScreenTape Assay (Agilent, 5067–5584, 5067–5585, 5067–5587, 5067–5603) on a 4200 TapeStation. Libraries were quantified using the Quant-iT PicoGreen dsDNA Assay (Thermo Fisher Scientific, P11496). Paired end 100 cycle sequencing was performed on a HiSeq 2500, HiSeq 4000, or NovaSeq 6000 System (all from Illumina) according to the manufacturer’s instructions.
7
Illumina-Based Validation Sequencing
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For sequencing of validation samples, Illumina’s next-generation sequencing methodology69 (link) was used. In detail, genomic DNA was quality-checked and quantified using the 4200 TapeStation instruments in combination with the Genomic DNA ScreenTape (both Agilent Technologies). Libraries were prepared from 100 ng of input material using Ovation RRBS Methyl-Seq with TrueMethyl oxBS (Tecan, Cat.no: 0553-32). In detail, only the bisulfite part of the protocol was followed, oxidation of DNA was not done, and samples were treated as MOCK oxBS samples according to the manufacturer’s instruction. Quantification and quality checks of libraries were done using the 4200 TapeStation and D5000 ScreenTapes instruments (both Agilent Technologies). Libraries were pooled and sequenced on a NovaSeq 6000 (Illumina) using S1 100-cycle reagents (Illumina, Cat.no: 20028319). The system was running in 101 cycle/single-end/standard loading workflow mode. Sequence information was converted to FASTQ format using bcl2fastq v2.20.0.422.
8
Transcriptome Profiling of Cortical Neurons
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Input samples were 400 primary cortical neurons from 4 WT biological replicates and 4 KO biological replicates. RNA was isolated using Zymo Quick-RNA Miniprep Plus Kit (R1057). Prior to library preparation, RNA integrity was confirmed using an Agilent 4200 TapeStation with high sensitivity RNA reagents (5067–5579). Sequencing libraries were prepared using the TruSeq Stranded mRNA kit (Illumina 20020595). Prior to sequencing, library size distribution was confirmed by capillary electrophoresis using an Agilent 4200 TapeStation with high sensitivity D1000 reagents (5067–5585), and libraries were quantified by qPCR using a KAPA Library Quantification Kit (Roche 07960140001). Libraries were sequenced on an Illumina NextSeq1000 instrument (66-bp read length, paired end).
9
Illumina Stranded Total RNA Sequencing
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Libraries were prepared using 50 ng of total RNA (all with RIN > 8.5) according to the manufacturer’s instructions with the Illumina® Stranded Total RNA Prep with Ribo-Zero Plus (Illumina, Inc., San Diego, CA, USA), including the depletion of cytoplasmic, mitochondrial and beta globin rRNA from 0.1–1 μg of intact total RNA samples. After rRNA depletion, libraries were prepared with 50 ng of total RNA (RIN > 8.5). Library integrity was confirmed by size analysis on a 4200 TapeStation (Agilent, Santa Clara, CA, USA) with a D1000 ScreenTape. All libraries were within an average size range of 360 to 400 bp. Library integrity and size (size range of 360 to 400 bp) was confirmed by analysis on a 4200 TapeStation (Agilent, Santa Clara, CA, USA) with D1000 ScreenTape. Library concentrations were determined using the dsDNA HS assay kit on Qubit 3.0 (Thermo Fisher Scientific, Waltham, MA, USA). Sequencing was performed with paired-end 75 cycles on a NextSeq 550 System (Illumina, Inc.) at the Instituto Tecnológico y de Energías Renovables.
10
Sequencing of Memory CD4+ T Cells
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RNA was extracted from FACS sorted target memory CD4+ T cells with RNeasy Micro Kit (Qiagen) according to the manufacturer's instructions. For preparation of RNA-Sequencing libraries, RNA concentration was measured using the Qubit RNA High Sensitivity kit (Life Technologies) and quality checked on the 4200 Tapestation using either the High Sensitivity or standard RNA ScreenTape assay (Agilent Technologies), depending on the measured RNA concentrations. PolyA-tailed mRNA was separated for sequencing during library preparation. Libraries were prepared using KAPA's mRNA HyperPrep kit (Roche Diagnostics) according to the manufacturer's instructions using an input of up to 200ng and a fragmentation incubation time of 8 minutes at 94°C. Samples were sequenced on Illumina's NextSeq500 (Illumina Cambridge) using a high output 75 cycle paired-end run. 24 libraries were multiplexed in the same run. Libraries were pooled in equimolar quantities, calculated from concentrations measured using the Qubit dsDNA High Sensitivity kit (Life Technologies) and fragment analysis using the D1000 High Sensitivity assay on the 4200 Tapestation (Agilent Technologies).
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