GSM2720063: ssMG-06d-22; Sus scrofa; RNA-Seq
Tissue was harvested and immediately frozen in liquid N2 and preserved at - 80°C until RNA extraction. Total RNA from 0.4 to 1.4 g of tissue was extracted with ice-cold Trizol reagent (Invitrogen, Carlsbad, CA). Tissue was weighed, placed in 10–15 ml of ice-cold TRIzol reagent, homogenized, and RNA extracted according to the manufacturer's protocol. RNA was resuspended in RNA storage buffer (Ambion, Austin, TX) and stored at −80°C until use. Quantity and purity of isolated RNA samples were analyzed with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). The RNA quality was assessed using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). The RNAseq libraries were prepared with Illumina's 'TruSeq Stranded mRNAseq Sample Prep kit' (Illumina). The libraries were quantitated by qPCR and sequenced on two lanes for 101 cycles from one end of the fragments on a HiSeq2500 using a HiSeq SBS sequencing kit version 4. Fastq files were generated and demultiplexed with the bcl2fastq v2.17.1.14 Conversion Software (Illumina). Adaptors have been trimmed from the reads with Trimmomatic (v0.33).