Genomic DNA was extracted from FFPE tissue with the AllPrep DNA/RNA FFPE kit (Qiagen, Hilden, Germany). Amplification and sequencing of the CDR3 of the different TCRβ families was performed using the ImmunoSeq™ (Adaptive Biotechnologies, Seattle, USA) protocol. In brief, highly optimized multiplexed PCR primers were used to amplify the respective CDR3s. Universal adaptor sequences and DNA barcodes were added by a second PCR run before high-throughput sequencing using the MiSeq ReagentKit v3 150-cycle in a MiSeq system (Illumina, San Diego, CA, USA).
Miseq reagent kit v3 150
Manufactured by Illumina
Sourced in United States
The MiSeq Reagent Kit v3 150 is a laboratory equipment product designed for use with the MiSeq sequencing platform. It provides the necessary reagents and consumables for performing 150-cycle sequencing runs on the MiSeq system.
Automatically generated - may contain errors
Lab products found in correlation
Miseq system, by Illumina (4 mentions)
Truseq rna sample prep kit v2, by Illumina (2 mentions)
2200 tapestation system, by Agilent Technologies (2 mentions)
Truseq small rna sample prep kit, by Illumina (2 mentions)
Immunoseq, by Adaptive Biotechnologies (1 mentions)
Allprep dna rna ffpe kit, by Qiagen (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Miseq ngs platform, by Illumina (1 mentions)
Agilent rna 6000 nano chip, by Agilent Technologies (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (1 mentions)
Nebnext library quant kit, by New England Biolabs (1 mentions)
Kapa library quantification kit, by Roche (1 mentions)
2100 bioanalyzer, by Illumina (1 mentions)
Nebnext multiplex small rna library prep set, by Illumina (1 mentions)
Miseq dna sequencer, by Illumina (1 mentions)
Miseq instrument, by Illumina (1 mentions)
Agilent rna 6000 nano kit, by Agilent Technologies (1 mentions)
Agilent dna 1000 kit, by Agilent Technologies (1 mentions)
Quantus fluorometer, by Promega (1 mentions)
7 protocols using miseq reagent kit v3 150
1
T-cell receptor sequencing of FFPE samples
2
Nanoparticle-Induced Transcriptomic Changes in Cancer-Associated Fibroblasts
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To investigate transcriptomic changes upon nanoparticle treatments in cancer-associated fibroblasts, co-culture experiments were performed. In the applied co-cultures, 4T1 cells were grown on the surface of 3.14 cm2 sized NUNC Polycarbonate Cell Culture Inserts, while NIH/3T3 cells were cultured below in the chambers of a 6 well plate. After 24 h of co-culturing, fibroblast cells were selectively treated with nanoparticles for 24 h, then RNA was purified from the fibroblast cells by Qiagen RNeasy mini kit. RNA concentrations were measured in a Qubit 2.0 fluorometer (Life Technologies), then RNA qualities were assessed in an Agilent 2100 Bioanalyzer by employing Agilent RNA 6000 nano Chip. Using 1 μg total RNA, non-strand specific sequencing library was prepared by Illumina TruSeq RNA sample Prep v2 kit, following the LS protocol. The size distribution of the generated library was assessed using DNA 1000 Chip in an Agilent 2100 Bioanalyzer, then the sequencing library was quantified by applying NEBNext Library Quant Kit (New England BioLabs) in a PikoReal real-time PCR system. Following quantification, 2 × 75 paired-end sequencing was performed on the samples using MiSeq Reagent kit V3-150 in an Illumina MiSeq NGS platform.
3
Small RNA-Seq Library Preparation and Sequencing
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For small RNA-Seq, 1 (one) μg of total RNA per sample was used for library preparation using TruSeq Small RNA Sample Prep Kits (Illumina, San Diego, CA, USA). Size-distribution was measured with the DNA ScreenTape assay on a 2200 TapeStation system (Agilent Technologies, US) and a real-time PCR with KAPA Library Quantification Kit (Kapa Biosystem, US) was used to quantify and evaluate the quality of each library. A total library pool of 4 nM was sequenced using a MiSeq Reagent Kit v3 150 cycle on a MiSeq System (Illumina, San Diego, CA, USA).
4
Small RNA Sequencing Protocol
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For small RNA-Seq, 1 μg of total RNA per sample was used for library preparation using TruSeq Small RNA Sample Prep Kits (Illumina, San Diego, CA, USA). Size-distribution was measured with the DNA ScreenTape assay on a 2200 TapeStation system (AgilentTechnologies, US). A total library pool of 4 nM was sequenced using a MiSeq Reagent Kit v3 150 cycle on a MiSeq System (Illumina, San Diego, CA, USA).
5
miRNA Sequencing Library Preparation
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miRNA sequencing libraries were prepared using NEBNext Multiplex Small RNA Library Prep Set for Illumina following the manufacturer’s protocol. Libraries were size selected using AMPure XP beads and after validation with an Agilent 2100 Bioanalyzer instrument sequenced with an Illumina MiSeq DNA sequencer using Illumina MiSeq reagent kit V3-150.
6
RNA Sequencing of MCF7 Ribosomes
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RNA integrity and quantity extracted from MCF7 ribosome pellets was determined by capillary gel electrophoresis using Agilent RNA 6000 Nano kit in an Agilent 2100 Bioanalyzer instrument. Poly-A selected, indexed RNA sequencing libraries (two biological replicates per treatment condition) were generated using TruSeq RNA Sample Prep Kit v2 (Illumina) following the protocol provided by the manufacturer. Purified sequencing libraries were validated and quantitated using Agilent DNA 1000 kit in an Agilent 2100 Bioanalyzer instrument. Sequencing libraries were pooled, denatured and sequenced in technical triplicates in an Illumina MiSeq instrument using MiSeq Reagent Nano Kit v2-500 and MiSeq Reagent Kit v3-150 kits (Illumina) generating 2×75 bp paired-end sequences.
FASTQ sequence files were generated by GenerateFASTQ 1.1.0.64 application on Illumina BaseSpace.
Adapters and low-quality sequences were trimmed with TrimGalore then reads were aligned to the Homo sapiens reference genome (GrCh38) using HISAT2. Gene specific read counts were determined with the summarizeOverlaps function of the Bioconductor R package using the GrCh38.104 transcriptome annotation.
FASTQ sequence files were generated by GenerateFASTQ 1.1.0.64 application on Illumina BaseSpace.
Adapters and low-quality sequences were trimmed with TrimGalore then reads were aligned to the Homo sapiens reference genome (GrCh38) using HISAT2. Gene specific read counts were determined with the summarizeOverlaps function of the Bioconductor R package using the GrCh38.104 transcriptome annotation.
7
Genome-wide Methylation Analysis of Murine Stromal Fibroblasts
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MBD-seq was performed to analyze genome-wide methylated and/or non-methylated DNA regions as previously described 23 (link) . Briefly, methylated DNA was enriched by methyl-CpG-binding domain protein 2 (MBD2)-mediated precipitation and subjected to next-generation sequencing. Highly methylated DNA regions were identified by sequence reads mapped on the reference genome. Extracted DNA from murine SF was sonicated with a Covaris sonicator to obtain ∼300 bp fragments. MBD2-mediated enrichment of methylated DNA was performed using the methylated DNA enrichment kit EpiXplore (Takara Bio) according to the manufacturer's instructions. The amount of enriched methylated DNA in 1 μg total DNA was measured using a Quantus Fluorometer (Promega, USA). Libraries for MBD-seq analysis were prepared using a QIAseq Ultralow Input Library Kit (Qiagen) according to the manufacturer's instructions and validated for an average size of ∼300-700 bp using a TapeStation and the Agilent High Sensitivity D1000 ScreenTape kit. Each experiment was biologically replicated at least three times. Sequencing of paired-end reads (75 bp) was performed using the MiSeq Reagent kit V3 150 cycle on a MiSeq system (Illumina) and mapped on the mouse genome (mm10) using CLC Genomics Workbench (QIAGEN).
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