Cellular RNA was purified using the AllPrep DNA/RNA kit (Qiagen, Ventura CA) as specified by the manufacturer. RNA quality was validated using the Agilent TapeStation, and the High Sensitivity RNA Screentape (Agilent, Santa Clara, CA) and quantity was determined using the Qubit 2·0 Fluorometer (ThermoFisher Scientific, Waltham, MA). cDNA was generated from 10 ng of total RNA using the SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Takara Bio USA, Inc., Mountain View, CA) according to manufacturer's directions. Then, 150 pg of the cDNA was used to prepare library for Illumina sequencing using the Nextera XT Library Prep Kit (Illumina, San Diego, CA) according to manufacturer's directions. Overall library size was determined using the Agilent TapeStation and the DNA High Sensitivity 5000 Screentape (Agilent, Santa Clara, CA). Equimolar number of libraries were pooled, denatured and High-Output, Single-Read, 36/38 base pair paired end Next Generation Sequencing was done on a NextSeq 500 (Illumina, San Diego, CA).
Agilent tapestation
Manufactured by Agilent Technologies
Sourced in United States, Australia, Denmark
The Agilent TapeStation is a lab equipment product designed for automated sample quality control and quantification. It utilizes microfluidic technology to analyze DNA, RNA, and protein samples in a rapid and efficient manner. The TapeStation provides users with accurate sample information, enabling informed decisions during the research or development process.
Automatically generated - may contain errors
Lab products found in correlation
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (46 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (14 mentions)
Nextseq 500, by Illumina (13 mentions)
Rneasy mini kit, by Qiagen (13 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (13 mentions)
Nanodrop, by Thermo Fisher Scientific (12 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (11 mentions)
Novaseq 6000, by Illumina (10 mentions)
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (9 mentions)
Trizol reagent, by Thermo Fisher Scientific (8 mentions)
Hiseq 2500, by Illumina (7 mentions)
Rneasy plus mini kit, by Qiagen (6 mentions)
Bcl2fastq2 v2, by Illumina (6 mentions)
Nextseq 550, by Illumina (6 mentions)
Trizol, by Thermo Fisher Scientific (6 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (6 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (6 mentions)
Recoverall total nucleic acid isolation kit, by Thermo Fisher Scientific (5 mentions)
Hiseq 4000, by Illumina (5 mentions)
Bcl2fastq2 v 2.17 program, by Illumina (5 mentions)
189 protocols using agilent tapestation
1
RNA Sequencing Library Preparation Protocol
2
RNA-Seq Library Preparation and Sequencing
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RNA was extracted using RNeasy Micro Kit (Qiagen) following the manufacturer’s instructions. The RNA samples were quantified using Qubit 2.0 Fluorometer (Invitrogen) and RNA integrity was checked with Agilent TapeStation (Agilent Technologies).
RNA sequencing libraries were prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina. Briefly, mRNA was first enriched with Oligod(T) beads. Enriched mRNAs were fragmented for 15 min at 94°C. First-strand and second-strand cDNA were subsequently synthesized. cDNA fragments were end repaired and adenylated at 3’ends, and universal adapters were ligated to cDNA fragments, followed by index addition and library enrichment by PCR with limited cycles. The sequencing library was validated on the Agilent TapeStation (Agilent Technologies) and quantified by using Qubit 2.0 Fluorometer (Invitrogen) as well as by quantitative PCR (KAPA Biosystems).
Libraries were sequenced on Illumina HiSeq 4000, using 2×150 bp paired end configuration, 50 million reads/sample. One mismatch was allowed for index sequence identification (Genewiz).
RNA sequencing libraries were prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina. Briefly, mRNA was first enriched with Oligod(T) beads. Enriched mRNAs were fragmented for 15 min at 94°C. First-strand and second-strand cDNA were subsequently synthesized. cDNA fragments were end repaired and adenylated at 3’ends, and universal adapters were ligated to cDNA fragments, followed by index addition and library enrichment by PCR with limited cycles. The sequencing library was validated on the Agilent TapeStation (Agilent Technologies) and quantified by using Qubit 2.0 Fluorometer (Invitrogen) as well as by quantitative PCR (KAPA Biosystems).
Libraries were sequenced on Illumina HiSeq 4000, using 2×150 bp paired end configuration, 50 million reads/sample. One mismatch was allowed for index sequence identification (Genewiz).
3
Transcriptomics of Sea Cucumber Development
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After testing the total RNA on an Agilent TapeStation (Agilent, USA), the libraries were prepared using a TruSeq Stranded mRNA Library Prep Kit (Illumina, USA), and fragments with a length of 200–450 nucleotides, including adapters, were selected. After testing the quality on an Agilent TapeStation, paired-end sequencing (2 × 100) was performed on an Illumina HiSeq2500. Raw reads were uploaded to the NCBI SRA database under the accession numbers SRR6075437, SRR6075438, SRR6075435, and SRR6075436 for the blastula, gastrula, auricularia, and pentactula stages, respectively.
4
Illumina and Nanopore Sequencing Libraries
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Genomic libraries for the short and long nucleotide reads were prepared following the manufacturer protocols for genome sequencing (Illumina HiSeq 2500 and PromethION Flow Cell, R9.4.1, Nanopore). The Illumina HiSeq 2500 method employed the NEBNext Ultra DNA Library Preparation Kit, with the subsequent quality assessment conducted using the Agilent TapeStation. In the Nanopore approach, native barcoding of the genomic DNA (EXP-NBD114 and SQK-LSk109) was explicitly executed for the PromethION system. Post-barcoding, stringent quality checks were performed using Qubit and the Agilent TapeStation to ensure the reliability and quality of the prepared libraries.
5
Total RNA Sequencing Library Preparation
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Total RNA samples were first evaluated for their quantity and quality using Agilent TapeStation. All the samples used for the sequencing had a RIN of at least 5. 100 ng of total RNA were used for library preparation with the KAPA total RNA Hyperprep Kit (KK8581) (Roche). Each resulting uniquely dual-indexed library was quantified and quality accessed by Qubit and Agilent TapeStation. Multiple libraries were pooled in equal molarity. The pooled libraries were sequenced on an Illumina NovaSeq 6000 sequencer with the v1.5 reagent kit. 100 bp paired-end reads were generated.
6
Illumina RNA-seq Library Preparation
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Library preparation and Illumina RNA-seq were performed as previously described (Mitra et al., 2021 (link)). Briefly, RNA samples were quantified using a Qubit 2.0 Fluorometer (ThermoFisher Scientific, Waltham, MA, United States) and RNA integrity was checked using an Agilent TapeStation (Agilent Technologies, Palo Alto, CA, United States). RNA sequencing libraries were prepared using the NEBNext Ultra II RNA Library Prep Kit for Illumina using manufacturer’s instructions (New England Biolabs, Ipswich, MA, United States). Sequencing libraries were validated on the Agilent TapeStation (Agilent Technologies, Palo Alto, CA, United States), and quantified with a Qubit 2.0 Fluorometer (ThermoFisher Scientific, Waltham, MA, United States) and with quantitative PCR (KAPA Biosystems, Wilmington, MA, United States). Sequencing libraries were multiplexed and clustered onto a flowcell, loaded onto the Illumina HiSeq instrument and sequenced using a 2 × 150bp Paired End configuration. Image analysis and base calling were performed using the HiSeq Control Software. Raw sequence data (.bcl files) generated from Illumina HiSeq was converted into fastq files and de-multiplexed using Illumina bcl2fastq 2.20 software. One mis-match was allowed for index sequence identification.
7
Total RNA Sequencing Workflow
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Total RNA samples were first evaluated for their quantity and quality using Agilent TapeStation. All the samples used for the sequencing had a RIN of at least 5. 100 nanograms of total RNA were used for library preparation with the KAPA total RNA Hyperprep Kit (KK8581) (Roche). Each resulting uniquely dual-indexed library was quantified and quality accessed by Qubit and Agilent TapeStation. Multiple libraries were pooled in equal molarity. The pooled libraries were sequenced on an Illumina NovaSeq 6000 sequencer with the v1.5 reagent kit. 100 bp paired-end reads were generated.
8
Comprehensive Transcriptome Analysis of hASC Differentiation
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Total RNA from in vitro differentiated hASC was subjected to quality control with Agilent Tapestation (Agilent Technologies Inc. Santa Clara, CA, USA) according to the manufacturer’s instructions. Libraries were constructed using Illumina TruSeq stranded mRNA sample preparation protocol according to manufacturer instructions (Illumina, San Diego, CA, USA). The yield and quality of the amplified libraries were analyzed using Qubit (Thermo Fisher Scientific) and the Agilent Tapestation (Agilent). The indexed cDNA libraries were normalized, combined, and the pools were sequenced on the Illumina Nextseq 550 (Illumina) for a 75-cycle v2 sequencing run generating 75bp single-end reads. Sample demultiplexing was performed using bcl2fastq (v2.20.0). Sample quality was assessed using FastQC (v0.11.8) and MultiQC (v1.7). Reads were aligned to a reference built from Ensembl GRCh38 genome sequences using STAR (v2.6.1d). Counts for each gene were obtained by featureCounts (v1.5.1) using the GRCh38.99 GTF file from Ensembl. Bioconductor package DESEq2 was used for count normalization and sample group comparisons, generating log2 fold changes, Wald test P values and P values adjusted for multiple testing (Benjamini-Hochberg method). RNAseq data are accessible on GEO with the accession number GSE164413 (reviewer token ctonmgksrjqpdwz).
9
Ribo-Zero rRNA Removal and Stranded mRNA Sequencing
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Ribosomal RNA was removed prior to library construction using Ribo-Zero rRNA Removal Kit Gram-Positive Bacteria (Illumina, MRZGP126). Stranded cDNA libraries were prepared using TruSeq Stranded mRNA Kit (Illumina, 20020594), quantitated by Agilent TapeStation, pooled, and sequenced on one flowcell lane for 75 cycles using paired-end 75 basepair sequencing on Illumina 2500 HiSeq Rapid Cluster Kit (Illumina, V2, PE-402-4002) and HiSeq Rapid SBS Kit (Illumina, V2, FC-402-4021).
10
Illumina RNAseq Library Preparation
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RNAseq library preparation was performed at Novogene Corporation utilizing the NEBNext Ultra RNA Library Prep Kit for Illumina by following the manufacturer’s recommendations (New England Biolabs, Ipswich, MA). Sequencing libraries were validated on the Agilent TapeStation (Agilent Technologies, Santa Clara, CA) and quantified using Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA) as well as by quantitative PCR (Applied Biosystems, Foster City, CA). The libraries were sequenced on an Illumina sequencer using a 2 × 150 Paired End configuration. Raw sequence data (.bcl files) was converted into fastq files and de-multiplexed using the Illumina’s bcl2fastq software.
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