Total RNA was extracted from root samples using TRIzol reagents (Tiangen Biotech Co., Ltd., China) according to the manufacturer's instructions. The RNAs were treated with RNase-free DNase I to eliminate contaminated genomic DNA. Two radish cDNA libraries, CK and Cr600, were constructed from control and 600 mg L−1 K2Cr2O7 treated root samples using the Illumina Paired End Sample Prep Kit. Briefly, poly (A) mRNA was enriched from total RNA using Sera-mag Magnetic Oligo (dT) Beads (Thermo Fisher Scientific, USA) and then mRNA-enriched RNAs were chemically fragmented to short pieces using the fragmentation solution (Ambion, USA). Double-stranded cDNA was generated using the Superscript Double-Stranded cDNA Synthesis Kit (Invitrogen, USA). After that, the Illumina Paired End Sample Prep Kit was used for RNA-seq library construction and was then sequenced using Illumina HiSeq™ 2000.
Paired end sample prep kit
Manufactured by Illumina
Sourced in United States, China
The Paired End Sample Prep kit is a laboratory equipment product designed to prepare DNA samples for paired-end sequencing. It provides the necessary reagents and protocols to generate sequencing libraries from DNA samples, enabling the analysis of both ends of DNA fragments. The core function of this kit is to facilitate the preparation of samples for paired-end sequencing applications.
Automatically generated - may contain errors
Lab products found in correlation
Hiseq 2000, by Illumina (9 mentions)
Superscript double stranded cdna synthesis kit, by Thermo Fisher Scientific (8 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (5 mentions)
Hiseq 2500 platform, by Illumina (4 mentions)
Microbexpress kit, by Thermo Fisher Scientific (4 mentions)
Clontech smarter ultra low rna kit, by Takara Bio (3 mentions)
Picogreen, by Thermo Fisher Scientific (3 mentions)
Hiseq 2000 platform, by Illumina (3 mentions)
Sureselect mouse all exon kit, by Agilent Technologies (3 mentions)
Rnase free dnase 1, by Thermo Fisher Scientific (3 mentions)
Hiseq 4000, by Illumina (3 mentions)
Nextseq 500, by Illumina (2 mentions)
Sera mag magnetic oligo dt beads, by Thermo Fisher Scientific (2 mentions)
Hiseq 2500, by Illumina (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Rna fragmentation kit, by Thermo Fisher Scientific (2 mentions)
Bacterial total rna extraction kit, by Merck Group (2 mentions)
Fragmentation solution, by Thermo Fisher Scientific (2 mentions)
Prime phase lock gel heavy tubes, by Thermo Fisher Scientific (2 mentions)
37 protocols using paired end sample prep kit
1
Transcriptome Profiling of Radish Root under Chromium Stress
2
Transcriptomic Profiling of Cochlear Sensory Epithelium
3
Transcriptome Analysis of Hainan Beech
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A C. hainanensis leaf transcriptome library was constructed using an mRNA-seq assay for paired-end Illumina sequencing, which was performed at Majorbio Biopharm Technology Co., Ltd. (Shanghai, China). Poly(A) mRNA was isolated from total RNA by using Sera-mag Magnetic Oligo (dT) Beads (Thermo Fisher Scientific, USA), and then mRNA-enriched RNAs were chemically fragmented to short pieces using the RNA Fragmentation Reagent (Ambion, USA). Double-stranded cDNA was synthesized using the SuperScript Double-Stranded cDNA Synthesis Kit (Invitrogen, Carlsbad, CA). Subsequently, the Illumina Paired End Sample Prep kit (Illumina, USA) was used to construct a RNA-seq library which then was sequenced by Illumina HiSeq 2000 (Illumina, San Diego, CA).
4
LCM RNA-Seq Workflow for Differential Gene Expression
5
Genomic Sequencing of African Swine Fever Virus
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Genomic DNA was extracted directly from low-volume ASFV PCR-positive porcine blood samples, designated G-2008/1 and G-2008/2, using the DNeasy Blood & Tissue kit (QIAGEN, Hilden, Germany). Village and district-level geographical locations were not available for the isolates. Genomic libraries were quantified using the Qbit (Qbit 2.0 Fluorometer, Life technologies) and sheared using the Covaris M220 focused ultrasonicator (Covaris, Inc.) Genome fragmentation was performed on the BioAnalyzer 2100 (Agilent Technologies, Inc.). Libraries were subsequently modified using the Illumina Paired End Sample Prep Kit and sequenced on the Illumina MiSeq platform (Illumina, Inc.) per manufacturer’s instructions. Raw fastq files for each virus sample were subjected to de novo assembly using CLCBio software (CLC Bio, Aarhus, Denmark). Mapped assemblies were also performed using the previously reported ASFV Georgia 2007/1 sequence (FR682468) as a reference. Variant calling, genome alignments and sequence illustrations were generated using Geneious software version 10.2.4. The C315R/C147L locus from global representatives were aligned using available nucleic acid sequence data from Genbank (Fig. 2 c).
6
Transcriptome Analysis of Fungal Mycelia
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Total RNA was extracted from mycelia using the TRIzol method (Invitrogen, Carlsbad, CA, United States), and the concentration and purity of the extracted RNA were detected by Nanodrop2000. RNA integrity was detected by agarose gel electrophoresis, and the RNA integrity number (RIN) value was determined by Agilent2100. The libraries were constructed after the RNA samples were qualified by using magnetic beads with Oligo(dT) and polyA to pair A-T bases to enrich mRNA. The fragmentation buffer solution was added to randomly break the mRNA into small fragments of about 300 bp. Double-stranded cDNA was synthesized using a SuperScript double-stranded cDNA synthesis kit (Invitrogen, Carlsbad, CA, United States) with random hexamer primers (Illumina). Then the synthesized cDNA was subjected to end-repair, phosphorylation, and “A” base addition according to Illumina’s library construction protocol. The cDNA library was constructed with an Illumina Paired End Sample Prep kit (Illumina, San Diego, CA, United States), quantified by TBS380 (Picogreen, Invitrogen, Carlsbad, CA, United States), and was then sequenced on the Illumina HiSeqTM2500 (2 × 150 bp read length) platform. The transcriptomeic data were analyzed online platform of Majorbio Cloud Platform (www.majorbio.com ).
7
RNA-Seq Library Preparation and Sequencing
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Total RNA was extracted from the flesh sample of each group via TRIzol Reagent (Invitrogen, USA) according to the manufacturer’s protocol. The quality and quantity of total RNA was assessed via NanoDrop2000 spectrophotometer (Thermo Fisher Scientific, USA), and RNA integrity was assessed on a 1.5% agarose gel. Purified poly (A) + mRNA was extracted from the total RNA sample using Oligo (dT) magnetic beads. mRNA was fragmented into 200–500 nt pieces by adding a fragmentation buffer. First-strand cDNA was synthesized using SuperScript II reverse transcriptase (Life Technologies, Inc.) and random hexamer primers. After generation of second-strand cDNA, the double-strand cDNA was end-repaired, and a single ‘A’ base and indexed adapters were ligated to the fragments. The cDNA library was constructed via the Illumina Paired End Sample Prep kit (Illumina, USA) and was then sequenced on the Illumina HiSeq. 2000 platform. Sequencing was conducted by the Majorbio Biopharm Technology Co. Ltd. (Shanghai, China). The quality of RNA-Seq was assessed via saturation, duplicate reads, and gene coverage analysis, using RSeQC-2.6.3 (http://rseqc.sourceforge.net/ ).
8
Transcriptomic Analysis of LCM Samples
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For LCM samples, next generation RNA sequencing was performed at University of Nebraska Medical Center Sequencing core. Briefly, synthesis of cDNA from 1-3ng of total RNA was performed with the Clontech SMARTer™ Ultra Low RNA Kit (Clontech Laboratories Inc, Mountain View, CA). Sequencing libraries were prepared using the Illumina Paired End Sample Prep Kit (Illumina Inc, San Diego, CA) according to Illumina's Ultra Low Input mRNA-Seq protocol. Each cDNA library was sequenced to generate 75 base pair reads, on the Illumina NextSeq500 (Illumina). The sequence reads were aligned to the mouse reference genome sequence (USCS mm10) using STAR aligner (Dobin et al., 2012) . Alignments were assembled and annotated using Cufflinks (Trapnell et al., 2010) . DESeq2 (Anders and Huber, 2010) was used to detect differentially expressed gene transcripts.
9
RNA-seq Analysis of Xanthomonas oryzae
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RNA-seq libraries were prepared using the Illumina Paired End Sample Prep kit as described previously (49 (link)). After removal of adaptors and low-quality reads, RNA-seq reads were aligned to the X. oryzae pv. oryzicola BLS256 genome using TopHat 2.0.7 (50 (link)), allowing for a maximum of two mismatched nucleotides. If reads mapped to more than one location in the genome, only the site showing the highest score was retained. Reads that mapped to tRNA or rRNA regions were removed; the remaining reads were mapped to the genome with HISAT2 (51 (link)) and used to generate a volcano plot. Bioconductor package edgeR (52 (link)) with TMM normalization was used to determine DEGs as described (13 (link)). Reproducibility was evaluated for two replicate experiments using pairwise linear correlation analysis prior to comparing RNA-seq profiles.
DEGs with significance (FDR < 0.01; fold change > 2) were selected for further analysis (differential expression data,https://drive.google.com/file/d/1FTiS4tQpVsyHcSvJKk4NtZZludVntRxE/view?usp=sharing ). Treeview 1.1.6 and Cluster 3.0 (53 (link), 54 (link)) were utilized to produce heatmaps based on reads per kilobase of transcript per million mapped reads (RPKM) (53 (link), 54 (link)).
DEGs with significance (FDR < 0.01; fold change > 2) were selected for further analysis (differential expression data,
10
Whole Exome Sequencing: Illumina Workflow
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Genomic DNA was obtained either from lymphocytes isolated from whole blood samples or from saliva using standard laboratory methods. The samples were prepared for whole exome sequencing (WES) using the Illumina paired-end sample prep kit (Illumina, San Diego, CA) and captured using the Agilent SureSelect Human All Exon Version 5 Enrichment kit (Agilent Technologies, Santa Clara, CA). The Agilent kit captures 96.5 megabases. Samples were sequenced at the Iowa Institute of Human Genetics Genomics Division using an Illumina HiSeq 2500, generating 125-bp paired-end reads. Subsequent data analysis was performed using the Argon computer cluster at the University of Iowa. FASTQs were mapped to the GRCh38 human reference sequence (Broad bundle, accessed at https://console.cloud.google.com/storage/browser/genomics-public-data/resources/broad/hg38/v0 ) using Burrows-Wheeler Aligner (BWA-MEM, version 0.7.10). Local realignment and base quality score recalibration were performed using Genome Analysis Toolkit (GATK) (http://www.broadinstitute.org/gatk ), and duplicate marking was performed using Picard tools (http:/picard.sourceforge.net). Haplotype caller was subsequently used to call genetic variants in standard Variant Call Format (vcf).
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