Truseq stranded total rna with ribo zero globin

For gene expression analysis, total RNA was collected using the mirVana RNA isolation kit (ThermoFisher Scientific) or RNeasy Mini Kit (Qiagen) from sorted (>20,000 cells) and bulk cultures (>1,000,000 cells). Illumina libraries were constructed using the TruSeq Stranded Total RNA with Ribo-Zero Globin (Illumina). Finally, libraries were quantified using Fragment Analyzer (Advanced Analytical). RNA-seq libraries were sequenced with HiSeq 4000 (Illumina) using a 2x76bp read length and alignment was performed using STAR Aligner^{92 (link)} against the GRCh38 reference genome. Gene counts were obtained using featureCounts^{93 (link)} and FPKM per gene were calculated using Cufflinks^{94 (link)}. Values were normalized using quantile normalization.

Total RNA was extracted from each sample (3 samples × 3 biological repeats) of D. nestor using Trizol reagents (Invitrogen, Carlsbad, CA, USA). The transcriptome library was constructed using the Illumina TruSeq Stranded total RNA with Ribo-Zero Globin (Illumina, San Diego, CA, USA), according to the manufacturer’s instructions. All cDNA libraries were subjected to high throughput sequencing using the Illumina HiSeq™ 4000 platform at Biomarker Technologies (Beijing, China). The raw sequencing data were cleaned and assembled using the Trinity software [32 (link)]. Gene expressions were normalized to the fragments per kilobase of transcript per million (FPKM) by HTSeq [33 (link)]. The expressions of genes were heatmapped and visualized using the Toolkit for Biologists, integrating various biological data handling tools [34 (link)].