GSM5005156: 28D4-IS; Ovis aries; RNA-Seq
Each sample, weighing approximately 50 mg, was finely minced and extracted in TRIzol (Thermo Fisher Scientific) by beads grinding using a TissueLyser LT (Qiagen) set at 50 oscillations per min (Qiagen). Two cycles of 10 min each were performed at 4°C and samples were visually inspected to ensure a complete homogenisation. RNA extraction was then carried out by chloroform precipitation and purified using RNeasy Mini Spin Columns (Qiagen). RNA sequencing library preparation used the NEBNext Ultra RNA Library Prep Kit for Illumina followed by manufacturer's instructions (New England Biolabs). Briefly, mRNA was first enriched with Oligod(T) beads. Enriched mRNAs were fragmented for 15 minutes at 94 ¡C. First strand and second strand cDNA were subsequently synthesized. cDNA fragments were end repaired and adenylated at 3'ends, and universal adapters were ligated to cDNA fragments, followed by index addition and library enrichment by PCR with limited cycles. The sequencing library was validated on the Agilent TapeStation (Agilent Technologies), and quantified by using Qubit 2.0 Fluorometer (Invitrogen) as well as by quantitative PCR (KAPA Biosystems). The sequencing libraries were clustered on one lane of a patterned flowcell. After clustering, the flowcell was loaded on the Illumina HiSeq 4000 or equivalent instrument in High Output Mode according to manufacturer's instructions. The samples were sequenced using a 2x150 paired end configuration. Image analysis and base calling were conducted by the HiSeq Control Software.