REFERENCE GENOME: ftp://ftp.ensembl.org/pub/release-75/fasta/ovis_aries/dna/Ovis_aries.Oar_v3.1.75.dna.toplevel.fa.gz
REFERENCE ANOTATION: ftp://ftp.ensembl.org/pub/release-75/gtf/ovis_aries/Ovis_aries.Oar_v3.1.75.gtf.gz
SAMPLES: available at https://www.ncbi.nlm.nih.gov/bioproject?LinkName=biosample_bioproject&from_uid=4252568
PROTOCOL: 
1. The read quality of the RNA-seq libraries was evaluated using FastQC 0.10.1 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). 
	fastqc Pairedend_1.fastq.gz
	fastqc Pairedend_2.fastq.gz
2. STAR (v.2.3.1y; https://github.com/alexdobin/STAR): Indexing Genome
	STAR \
		--runMode genomeGenerate \
		--genomeDir /PATHtoReferenceGenome \
		--genomeFastaFiles /PATHtoReferenceGenome/Ovis_aries.Oar_v3.1.75.dna.toplevel.fa \
		--runThreadN 8
3. STAR (v.2.3.1y; https://github.com/alexdobin/STAR): Mapping reads against the ovine genome assembly v.3.1. (Oar_v3.1) 
	STAR \
      		--genomeDir /PATHtoReferenceGenome \
      		--readFilesIn sampe_1.fastq.gz sample_2.fastq.gz \
      		--readFilesCommand zcat \
      		--runThreadN 8 \
      		--outSAMstrandField intronMotif
4. Samtools (v.0.1.19; https://sourceforge.net/projects/samtools/files/samtools/0.1.19): sorting the alignment files
	samtools view -Sb -@ 4 sample.sam | samtools sort -@ 4 sample.sort.bam
5. Samtools (v.0.1.19; https://sourceforge.net/projects/samtools/files/samtools/0.1.19): removing duplicates
	samtools rmdup -S sample.sort.bam sample.sort.rmdup.bam 
6. Samtools (v.0.1.19; https://sourceforge.net/projects/samtools/files/samtools/0.1.19): merging “rmdup.bam” files
	samtools merge -@ 8 merged.bam sample1.sort.rmdup.bam sample2.sort.rmdup.bam … sampleN.sort.rmdup.bam
7. Alignment with Cufflinks (Cufflinks v.2.1.1; http://cole-trapnell-lab.github.io/cufflinks/)
	cufflinks \
      		--output-dir MERGE \
      		--num-threads 12 \
      		-g Ovis_aries.Oar_v3.1.75.gtf \
      		--max-intron-length 50000 \
		merged.bam
##“- g” followed by the gtf file from the Oar_v3.1 reference sequence was used to guide the assembly but not excluding new genes
8. Cuffmerge (Cufflinks v.2.1.1; http://cole-trapnell-lab.github.io/cufflinks/)
	echo “$PATHtoMERGE/transcripts.gtf” > list.txt
	cuffmerge -p 8 -o CUFFMERGE - g Ovis_aries.Oar_v3.1.75.gtf list.txt
## Cuffmerge was used to filter genes with low or no expression from our reference gtf file.
9. SigCufflinks (available at http://www.sigenae.org) was used to obtain raw counts per gene, we used as reference for the quantification the annotation “gtf” file from cuffmerge step.
	sigcufflinks -o sample.counts -t 8 -G CUFFMERGE/merged.gtf -q sample.sort.bam
	paste sample1.counts sample2.counts … sampleN.counts |cut -f1,2…N > GSE74825_Procesed_global_count_genes_milk.csv
## GSE74825_Procesed_global_count_genes_milk.csv available at ftp://ftp.ncbi.nlm.nih.gov/geo/series/GSE74nnn/GSE74825/suppl/GSE74825_Procesed_global_count_genes_milk.csv.gz