RNA samples that pass quality parameters (minimum concentration and size range) were used to develop RNA libraries using the TruSeq Stranded Total RNA with Ribo-Zero Kit, Set A (FC-122–2501, San Diego, CA) according to manufacturer’s instructions. Each sample is prepared using 1 µg total RNA. The resulting cDNA libraries were quantified using the Qubit system (Invitrogen, Carlsbad, CA) and checked for quality and size using the Experion DNA 1K chip (BioRad, Hercules, CA). The fragment size generated library was ranging from 220 to 500 bps with a peak at ~250 bps. A portion of each library was diluted to 10 nM and stored at −20°C. 10 µL of 1.2–1.8 nM libraries was diluted and denatured. The libraries were sequenced using NextSeq500 High Output Kit (300 cycles- PE100) on Illumina NextSeq500 platform. The sequencing reads were automatically uploaded and evaluated for quality control using Illumina BaseSpace Onsite Computing platform.
Experion dna 1k chip
Manufactured by Bio-Rad
Sourced in United States
The Experion DNA 1K chip is a lab equipment product designed for the analysis and quantification of DNA samples. It can process up to 12 samples simultaneously and provides information on the size and concentration of DNA fragments ranging from 25 to 1,000 base pairs.
Automatically generated - may contain errors
Lab products found in correlation
Nextseq 500 platform, by Illumina (5 mentions)
Qubit system, by Thermo Fisher Scientific (5 mentions)
Nextseq500 high output kit 300 cycles pe100, by Illumina (4 mentions)
Basespace onsite computing platform, by Illumina (4 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (1 mentions)
Nextseq 500 high output kit, by Illumina (1 mentions)
Truseq total stranded rna with ribozero kit, by Thermo Fisher Scientific (1 mentions)
Hiseq 2500, by Illumina (1 mentions)
Dnase digestion, by Merck Group (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Spectrum plant total rna kit, by Merck Group (1 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (1 mentions)
Trueseq rna sample prep kit, by Illumina (1 mentions)
6 protocols using experion dna 1k chip
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RNA-Seq Library Preparation and Sequencing
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Transcriptomic Profiling of IL-4-treated Macrophages
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Transcriptomic Profiling of IL-4-treated Macrophages
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To determine the effect of IL-4 on d3 MI macrophages, whole-transcriptome analysis by RNA sequencing (RNA-seq) was performed [25 ,26 (link)]. Sample quality was evaluated and used to develop RNA libraries (n = 21 pooled index samples) using the TruSeq Stranded Total RNA with Ribo-Zero Kit, Set A (FC-122–2501, San Diego, CA) following manufacturer protocol. The cDNA libraries obtained were assessed using the Qubit system (Invitrogen, Carlsbad, CA) and examined for quality using Experion DNA 1 K chip (BioRad, Hercules, CA). The libraries were sequenced using NextSeq 500 High Output Kit (300 cycles-PE100) on the Illumina NextSeq 500 platform and the reads were checked for quality using Illumina BaseSpace Onsite Computing platform. A total of 24,341 genes were sequenced, of which there were two sets of duplicates, giving a total of 24,339 analyzed (Supplemental Table 1 ). Validation was performed by qRT-PCR for Il4, Tgfb1, Arg1, Ym1, Pdgfc, and Fam198b, with comparisons made by regression analysis (Supplemental Fig. 3 ). Visualization tools provided in Metaboanalyst 3.0 (http://www.metaboanalyst.ca ) were used to analyze clustering and correlation. Enrichment analysis for significantly different proteins was performed using Enrichr (http://amp.pharm.msm.edu/Enrichr/ ) gene ontology (GO) biological processes.
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RNA-Seq Library Preparation and Sequencing
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RNA was extracted using the Pure Link RNA Mini Kit (Ambion; Foster City, CA, USA) according to the manufacturer’s instructions, and its quality was assessed based on the minimum concentration and size range. The cDNA libraries were established using the TruSeq Total Stranded RNA with RiboZero Kit (Ambion, Set-A) and quantified using the Qubit System (Invitrogen; Carlsbad, CA, USA), and the quality and size were assessed using the Experion DNA 1 K Chip (Bio-Rad; Hercules, CA, USA). All the samples surpassed the quality control standards of minimum concentration and RNA quality indicator > 9, as demonstrated by discrete 18S and 28S bands. The library of n = 6 pooled samples was sequenced using the NextSeq 500 High Output Kit (300 cycles, paired-end 100 bp) with the Illumina NextSeq 500 platform (Illumina; San Diego, CA, USA). The sequenced reads and Fastq sequence files were used to align reads to the reference genome USCS-GRCm38/mm10 in the RNA-Seq Alignment Application using STAR aligner with the Illumina Cloud Computing Platform. The fragments per kilobase of transcript per million mapped reads (FPKM) values of the reference genes and transcripts were generated using Cufflinks 2.
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Transcriptome Profiling of Leaf Tissues
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Leaf tissue samples (100 mg) for RNA isolation were collected from each genotype at 0, 24 and 48 h post inoculation (hpi). Total RNA was isolated as described in Spectrum™ Plant Total RNA kit protocol with on-column DNase digestion (Sigma-Aldrich, USA). RNA samples were purified using RNA clean and concentration TM-25 (ZYMO RESEARCH) and the quality was determined both by NanoDrop and Agilent 2100 Bioanalyzer. A total RNA (~ 3 μg) for each sample was used to prepare mRNA-seq library according to TrueSeq RNA Sample Prep Kit protocol (Illumina). Library quality control and quantification were performed with an Experion DNA 1 K Chip (Bio-Rad) and a Qubit fluorometer (Invitrogen), respectively. For each library, more than 100 million, 100-bp paired-end sequences were generated (Table 1 ) using an Illumina HiSeq 2500.
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RNA-Seq Library Preparation and Sequencing
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