Adult_2 transcriptome Artyfechinostomum sufrartyfex
Total RNA was extracted from adult lifecycle stage (duplicate2) using Trizol reagent following manufacturers protocol. Library layout fixed was paired-end. After performing quality control (QC), passed samples were taken forward for library construction. The RNA samples with an RNA integrity number (RIN) of 7.0 and above was used for mRNA purification. mRNA was purified using oligo-dT beads (TruSeq RNA Sample Preparation Kit, Illumina) taking 1 g of intact total RNA. The purified mRNA was fragmented at 90C 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 Agencourt Ampure XP SPRI beads. The sequencing library was prepared by random fragmentation of the cDNA sample, followed by 5' and 3' adapter ligation, after end-repair and the addition of an A base and SPRI cleanup. The prepared cDNA library was amplified using PCR for the enrichment of the adapter-ligated fragments. The library was then quantified using a NanoDrop spectrophotometer (Thermo Scientific) and validated for quality with a Bioanalyzer (Agilent Technologies). Adapter-ligated fragments were then PCR amplified and gel purified. For cluster generation, the library was loaded into a flow cell. Each fragment was then amplified into distinct, clonal clusters through bridge amplification. When cluster generation was complete, the templates were sequenced utilizing Illumina SB