Subclinical Mastitis_1
RNA sequencing libraries were prepared with Illumina-compatible NEBNext Ultra II Directional RNA Library Prep Kit at Genotypic Technology Pvt. Ltd., Bangalore, India. 100-500 ng of total RNA was taken for mRNA isolation, fragmentation and priming. Fragmented and primed mRNA was further subjected to first strand synthesis followed by second strand synthesis. The double stranded cDNA was purified using NEBNext sample purification beads. Purified cDNA was end-repaired, adenylated and ligated to Illumina adapters as per NEBNext Ultra II Directional RNA Library Prep protocol followed by second strand excision using USER enzyme at 37 C for 15mins. Adapter ligated cDNA was purified using NEBNext beads and was subjected to 11-14 cycles for Indexing-(98C for 30 sec, cycling (98C for 10sec, 65C for 75sec) and 65C for 5min) and enrich the adapter-ligated fragments. Final PCR products (sequencing library) were purified with NEBNext beads, followed by library quality control check. Illumina-compatible sequencing library were quantified by Qubit fluorometer and fragment size distribution was analyzed on Agilent 2200 TapeStation.