RNA-seq of ovis aries: amniotic epithelial cells
RNA extraction was performed on each biological sample using RNeasy Mini Kit (QIAGEN) according to the manufacturers instruction. RNA was quantified and checked for RNA integrity number (RIN) using the Agilent 2100 Bioanalyzer imposing a minimum value of 8.0 for processing. mRNA (poly-A) was isolated using oligo-dT beads (Illumina) then purified using mRNA purification kit (Invitrogen). After the purification step, RNA was fragmented and was copied-back into first strand cDNA using SuperScript II (Invitrogen) and random primers (Illumina). The second strand of cDNA was synthesized by adding buffer dNTPs, DNA Polymerase I and RNase H. Following adenylation of 3 ends of cDNA fragments, Illumina adapters were ligated to prepare for hybridization. The sequencing reaction has been performed on an Illumina HiSeq 2500 platform in single-end for 75 cycles ensuring at least 30 million of reads per sample. To assess the quality of sequences, raw reads were subjected to quality check using fastQC tool (https://www.bioinformatics.babraham.ac.uk/projects/fastqc) and then trimmed by Trimmomatic software, imposing a minimum quality per base of 24 and a minimum length of reads equal to 36 bases (ILLUMINACLIP:TruSeq3-SE:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:24 MINLEN:36). Transcriptome sequencing returned from 28.2 to 35.2 million of clean reads with an alignment rate, calculated by Kallisto aligner against the reference transcriptome (OviAries 3.1 sheep genome, Ensembl 99), ranging from 44.8% to 49.1% reporting a large amount of not annotated transcripts.