RNA was extracted from 50,000 FACS-purified CD8+ T cells per subset using a Direct-Zol RNA Microprep Kit (Zymo Research) and stored at −80°C. Quality control was performed using a High Sensitivity RNA ScreenTape Assay with a 4200 TapeStation System (Agilent). Libraries for mRNA sequencing were prepared from 5 ng of total RNA using the SMART-Seq v4 Ultra Low Input RNA Kit (Clontech-Takara). Full-length cDNAs were processed using a Nextera XT DNA Library Preparation Kit (Illumina). Quality control was performed using a High Sensitivity DNA ScreenTape Assay with a 4200 TapeStation System (Agilent). Libraries were then multiplexed in an equimolar pool and sequenced using a NextSeq 500/550 Platform (Illumina). An average of 11 million single-end 75 base pair (bp) reads were generated per sample. Libraries for total RNA sequencing were prepared from 1 ng of total RNA using a SMART-Seq Stranded Kit (Clontech-Takara). Quality control and sequencing were performed as described for the mRNA libraries, generating an average of 103 million paired-end 75 bp reads per sample.
4200 tapestation system
Manufactured by Agilent Technologies
Sourced in United States
The 4200 TapeStation System is a compact, automated instrument designed for the quality control and sizing of nucleic acid samples. It utilizes microfluidic-based electrophoresis technology to provide reliable and reproducible results for a wide range of sample types, including DNA, RNA, and PCR amplicons.
Automatically generated - may contain errors
Lab products found in correlation
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (25 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (15 mentions)
Nextseq 500, by Illumina (14 mentions)
Novaseq 6000, by Illumina (14 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (13 mentions)
Rneasy mini kit, by Qiagen (12 mentions)
D1000 screentape, by Agilent Technologies (11 mentions)
Nextera xt index kit, by Illumina (10 mentions)
Ampure xp bead, by Beckman Coulter (10 mentions)
Trizol reagent, by Thermo Fisher Scientific (10 mentions)
Miseq platform, by Illumina (9 mentions)
Chromium controller, by 10x Genomics (7 mentions)
Nextseq 500 system, by Illumina (7 mentions)
Kapa library quantification kit, by Roche (7 mentions)
Chromium next gem chip g, by 10x Genomics (6 mentions)
Chromium next gem single cell 3 reagent kits v3, by 10x Genomics (6 mentions)
Hiseq 2500, by Illumina (6 mentions)
Hiseq 4000, by Illumina (6 mentions)
Nextera xt kit, by Illumina (6 mentions)
Nanodrop one, by Thermo Fisher Scientific (6 mentions)
246 protocols using 4200 tapestation system
1
Transcriptome Profiling of CD8+ T Cells
2
Transcriptome Profiling of CD8+ T Cells
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RNA was extracted from 50,000 FACS-purified CD8+ T cells per subset using a Direct-Zol RNA Microprep Kit (Zymo Research) and stored at −80°C. Quality control was performed using a High Sensitivity RNA ScreenTape Assay with a 4200 TapeStation System (Agilent). Libraries for mRNA sequencing were prepared from 5 ng of total RNA using the SMART-Seq v4 Ultra Low Input RNA Kit (Clontech-Takara). Full-length cDNAs were processed using a Nextera XT DNA Library Preparation Kit (Illumina). Quality control was performed using a High Sensitivity DNA ScreenTape Assay with a 4200 TapeStation System (Agilent). Libraries were then multiplexed in an equimolar pool and sequenced using a NextSeq 500/550 Platform (Illumina). An average of 11 million single-end 75 base pair (bp) reads were generated per sample. Libraries for total RNA sequencing were prepared from 1 ng of total RNA using a SMART-Seq Stranded Kit (Clontech-Takara). Quality control and sequencing were performed as described for the mRNA libraries, generating an average of 103 million paired-end 75 bp reads per sample.
3
Transcriptome Analysis of Tomato Immunity
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Total RNA was isolated with the RNeasy Plant Mini Kit (QIAGEN) from wild-type and fir1-1 tomato leaves inoculated by vacuum with P. fluorescens A506 or a mock solution (three biological replicates for treatment, in total 12 samples collected at 6 h postinoculation). RNA integrity was evaluated using the 4,200 TapeStation System (Agilent Technologies). RNA-Seq cDNA libraries were prepared from RNA samples using the NEBNext Ultra ™ II mRNA Library Prep Kit for Illumina and then PCR-amplified using NEBNext Multiplex Oligos for Illumina (New England Biolabs). Quality and average size of cDNAs in the library were evaluated using the 4,200 TapeStation System (Agilent Technologies).
4
Transcriptome Profiling of Whole Blood
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Patient blood from cohort one was sampled into PAXgene Blood RNA Tubes (Qiagen) and RNA extraction was performed following manufacturer’s instructions using either the PAXgene Blood RNA Kit or the QIAsymphony PAXgene Blood RNA Kit on a QIAsymphony SP automated liquid handling system (Qiagen). In both cases RNA integrity and quantity was determined via RNA assay on a Tapestation 4200 system (Agilent). 750ng total RNA were subsequently used as an input for NGS library prep according to the TruSeq Stranded Total RNA with Ribo-Zero Globin kit (Illumina) protocol using IDT for Illumina TruSeq UDI adapters (Illumina). Libraries were quantified using the Qubit HS dsDNA assay (Thermofisher) and library fragment size distribution was determined using the D1000 assay on a Tapestation 4200 system (Agilent). Libraries were equimolarly pooled into pools of 96 samples and clustered lane wise at 200p.m. concentration on NovaSeq6000 S2 flow cells. Raw sequencing data was demultiplexed and processed into fastq files using bcl2fastq2 v2.20 and subsequently aligned to human genome build GRCh38 using STAR aligner. Reads were sequenced paired-ended for 50 bp each with an average depth of 2.4×107 reads per sample, with a mean alignment rate (unique and multiple mappings) of 89.4%.
5
RNA-seq Analysis of C. elegans
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Adult nematodes were dissolved in 1 ml Trizol (Invitrogen) and total RNA was isolated via the miRNeasy Micro kit (Qiagen) according to the manufacturer's protocol. RNA concentration and integrity were determined by High Sensitivity RNA assay on a TapeStation 4200 system (Agilent). cDNA libraries were prepared from 5 ng total RNA using the SMART-seq2 protocol and tagmented with the Nextera XT kit (Illumina). Library purification and size selection was carried out with AMPure XP beads (Beckman–Coulter) and final library size distribution was measured via High Sensitivity D5000 assay on a TapeStation 4200 system (Agilent). Library concentration was determined using the HS dsDNA assay on a Qubit 3. Libraries were sequenced SR 75 cycles on a NextSeq500 system (Illumina) using High Output v2 chemistry. Base call files were converted to fastq format and demultiplexed using bcl2fastq v2.20. The 75 bp single-end reads were aligned to the C. elegans reference transcriptome WBcel235 by kallisto v0.44.0 using default parameters. Raw RNA seq data were deposited to GEO database under the common accession number GSE197286 .
6
Smart-Seq2 Protocol for Single-Cell RNA-Seq
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Individual lysed cells were treated according to the Smart-Seq2 library preparation protocol.64 (link) Preamplified cDNA was quantified and the average size distribution was determined via D5000 assay on a tapestation4200 system (Agilent). Tagmentation and subsequent next-generation sequencing (NGS) library generation were performed using 200 pg of cDNA per sample following the Smart-Seq2 protocol. NGS libraries were quantified by HS dsDNA assay on a Qubit (Invitrogen) and the average size distribution was determined via D5000 assay on a tapestation4200 system (Agilent). Libraries were equimolarly pooled, clustered at 1.4 pM, and sequenced using SR 75 cycles High Output v2 chemistry on a NextSeq500 system (Illumina). Raw sequencing data were demultiplexed and converted into fastq format using bcl2fastq2 v2.20 and aligned to mouse genome M16 via STAR aligner considering both, exonic and intronic reads.
7
Insect RNA-Seq Library Preparation
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Total RNA was isolated from the insect sample in TRIzol using Direct-Zol R.N.A. Miniprep Kit (ZYMO Research). The quality and quantity of the isolated RNA were analyzed using a 1.2% agarose gel and NanoDropTM 1000 (Thermo Fisher Scientific, Wilmington, DE, USA) reading, respectively. Paired-end RNA-Seq libraries were prepared using an Illumina TruSeq stranded mRNA library preparation kit. The fragments of Poly A-tailed mRNA were pulled out from the total RNA using poly-T attached magnetic beads and subjected to enzymatic fragmentation. Following the synthesis of the first strand of cDNA, the RNA-dependent synthesis of the second strand was facilitated using a strand and ACTIN-D mix. The double-stranded cDNA was then purified with XP (Ampure) beads, followed by adapter ligation, A-tailing, and a limited number of PCR cycles to enrich it. The PCR-enriched libraries were analyzed for quality and quantity in a 4200 TapeStation system (Agilent Technologies, Santa Clara, CA, USA) using High sensitivity D1000 Screen tape. The paired-end (PE) libraries were loaded onto NextSeq 500 (Illumina, San Diego, CA, USA) for cluster generation and sequencing (2 × 75 bp).
8
Intestinal Transcriptome Analysis of Newborn Offspring
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On postnatal day 1 offspring were euthanized, the entire gastrointestinal tract was excised, and the ileum was rapidly frozen in liquid nitrogen and maintained in –80 °C until RNA isolation. For bulk RNA sequencing analysis, tissues were homogenized in Trizol, and messenger RNA was purified using the RNeasy Mini kit (Qiagen). Illumina single-end cDNA libraries of postnatal day 1 ileum mRNA was prepared from 250 ng total RNA using the TruSeq Stranded mRNA Kit with poly-A enrichment (RS-122-2101, Illumina) according to the manufacturer’s protocol. Library fragment size was quantified using an Agilent High Sensitivity D1000 ScreenTape Assay on an Agilent 4200 Tapestation System (G2991AA). Library concentration was quantified using the Qubit dsDNA HS (High Sensitivity) Assay Kit. Samples representative of all treatment groups were multiplexed and sequenced on an Illumina NextSeq500 instrument using high-output 1 × 75 bp geometry (Illumina).
9
RNA Extraction and RT-PCR Analysis
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Total RNA was extracted using the Direct-zol RNA MiniPrep (Zymo Research). cDNA was synthesized using SuperScript IV Reverse Transcriptase (Thermo Fisher Scientific) following the manufacturer’s protocol. RT-PCR was performed using gene specific primers (Supplementary Table 7 ) using Q5 High-Fidelity DNA Polymerase (New England Biolabs) and amplicons were analyzed and quantified using either an 4200 TapeStation System (Agilent Genomics) or agarose gel electrophoresis followed by quantification of band intensity using FIJI/ImageJ. To detect poison exon-containing RNA isoforms, cells were treated with 50 μg/mL cycloheximide for up to 6 hours to inhibit NMD.
10
Total RNA Extraction from Cells
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