Expression analysis of Water buffalo (Bubalus bubalis) associated with economically important traits
Total RNA was extracted from four different groups of Bubalus bubalis. The quality and quantity of the extracted RNA was analyzed using an Agilent 2100 Bioanalyzer (Agilent Technologies). The extracted RNA with an RNA integrity number of 8.0 was used for mRNA purification. mRNA was purified using oligo-dT beads (TruSeq RNA Sample Preparation Kit, Illumina) taking 1 micro gram of intact total RNA. The purified mRNA was fragmented at 90 degree C in the presence of divalent cations. The fragments were reverse transcribed using random hexamers and Superscript II Reverse Transcriptase (Life Technologies). Second strand cDNA was synthesized on the first strand template using RNaseH and DNA polymerase I. The cDNAs so obtained were cleaned using Beckman Coulter AgencourtAmpure XP SPRI beads. The prepared cDNA library was amplified using PCR for the enrichment of the adapter-ligated fragments. The individual libraries were quantified using a NanoDrop spectrophotometer (Thermo Scientific) and validated for quality with a Bioanalyzer (Agilent Technologies). These were then sequenced using the IlluminaHiSeq 2500 platform. Paired-end FASTQ files were subjected to standard quality control based on Phred scores greater than 20 using the NGSQC Tool Kit to obtain high quality (HQ) filtered reads.