title: GSM6588585: ART_#4 Kidney; Bos indicus x Bos taurus; RNA-Seq
library_construction_protocol: Total RNA was isolated using TRIzol™ Reagent (Invitrogen, 15596026) following the manufacturer's instructions. The concentration of RNA samples was measured by using the NanoDrop® ND-1000 Spectrophotometer. RNA samples were stored at -80°C. This RNA-seq was conducted by Novogene (Sacramento, CA, USA). Briefly, total RNA samples were shipped on dry ice to Novogene overnight. Quality controls were performed for all samples using agarose gel electrophoresis to test RNA degradation and potential contamination and using Agilent 2100 Bioanalyzer to check RNA integrity and quantitation. All samples showed RNA integrity numbers (RIN) of 7.5 to 9.1 and passed the quality control. For strand specific sequencing library preparation, mRNA was enriched using oligo(dT) beads and fragmented randomly using fragmentation buffer. cDNA was synthesized by using mRNA template and random hexamers primer, after which a custom second-strand synthesis buffer (Illumina), dNTPs (dUTP instead of dTTP), RNase H and DNA polymerase I were added to initiate the second-strand synthesis. Then, double-stranded cDNA was subjected to terminal repair, 3' adenylation, sequencing adaptor ligation, uracil-DNA glycosylase degradation, size selection for 250-300bp insert, and PCR enrichment. Quality controls were performed for all libraries including Qubit concentration measurement, testing insert size using Agilent 2100 Bioanalyzer, and quantifying effective concentration using qPCR. Qualified libraries were pooled and sequenced on Illumina HiSeq platform for 150bp paired end reads.