Truseq stranded total rna with ribo zero gold

RNA sequencing was performed by the Shanghai OE Biotech (Shanghai, China). In brief, the mirVana miRNA Isolation Kit (Ambion) was used to extract the total RNA from liver tissues. The TruSeq Stranded Total RNA with Ribo-Zero Gold (Illumina) was used to construct cDNA libraries. The purified cDNA libraries were sequenced on Illumina HiSeq 2500 following the manufacturer's instruction. After filtrating the adaptor and low-quality reads, clean reads were obtained for subsequent analysis. The reads were matched to the rat reference genome using the hisat2 (v2.2.1.0) software. The StringTie2 (v1.3.3b) software was used to splice the aligned reads. lncRNA identification included two categories: one is known lncRNA, which completely matches with the known lncRNAs, and the other is candidate lncRNA lacking protein-coding ability, whose length is greater than 200 bp and exon is greater than or equal to 2. The software CPC (v0.9-r2), CNCI (v1.0), Pfam (v30), and PLEK (v1.2) were used to predict the protein-coding ability of transcripts. The expression of transcription was calculated by the fragments per kilobase of exon per million reads mapped (FPKM) method using the bowtie2 (v2.2.9) and eXpress (v1.5.1) software.

A global analysis of pulmonary arterial ECM gene expression was performed by total RNA sequencing and subsequent analysis of matrisome genes. Snap-frozen PA specimens were subjected to TRIzol-RNA extraction. RNA quality was assessed by measuring the RNA integrity number (RIN) using a Fragment Analyzer HS Total RNA Kit (Advanced Analytical Technologies, Inc.). Library preparation for total RNA-Seq was performed using TruSeq™ Stranded Total RNA with Ribo-Zero Gold (Illumina, Cat. Nr. RS-122-2301) starting from 1000 ng of total RNA. The size range of the final cDNA libraries was determined by applying the SS NGS Fragment 1- to 6000-bp Kit on the Fragment Analyzer (average 340 bp). Accurate quantification of cDNA libraries was performed by using the QuantiFluor™ dsDNA System (Promega). cDNA libraries were amplified and sequenced by using HiSeq 2000 from Illumina^{112 (link)}.

We collected 15 Co. inflata individuals on Friday Harbor, WA, on August 8–15, 2015, brought them back to Friday Harbor Lab, and allowed them to spawn in a sea-table. We pooled a wide range of embryonic stages along with hatched larvae in Eppendorf tubes, pipetted vigorously to remove follicle cells, allowed the embryos and larvae to settle, and then rinsed them in 500 μl of 0.2-μm filtered seawater. The tubes were spun down at 3,000 rpm for 1 min, excess water was removed, and samples were frozen in liquid nitrogen and stored at −80 °C until RNA isolation. All samples were pooled and total RNA was isolated using the Qiagen RNeasy Lipid Tissue Mini Kit and treated with DNAase. We checked RNA quality on an Agilent bioAnalyzer chip and sent the RNA to the University of Pennsylvania Next Generation Sequencing Core, where a library was generated using Illumina TruSeq Stranded Total RNA with Ribo Zero Gold. This library was sequenced using an Illumina HiSeq 2500 to generate 100-bp paired-end reads.