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.
Nextseq500 high output kit 300 cycles pe100
Manufactured by Illumina
The NextSeq 500 High Output Kit (300 cycles - PE100) is a laboratory equipment product designed for sequencing applications. It provides a kit with the necessary reagents and consumables to perform up to 300 cycles of paired-end sequencing at 100 base pairs per read on the NextSeq 500 sequencing system.
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4 protocols using nextseq500 high output kit 300 cycles pe100
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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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