RNA-seq of Prionotus carolinus: proximal half of leg : replicate 2
Total RNA was extracted from frozen cell samples using Qiagen RNeasy Plus Universal mini kit following manufacturers instructions (Qiagen, Hilden, Germany). RNA samples were quantified using Qubit Fluorometer (Life Technologies, Carlsbad, CA, USA) and RNA integrity was measured using the RNA Screen Tape on Agilent 2200 TapeStation (Agilent Technologies, Palo Alto, CA, USA). The RNA sequencing libraries were prepared using the NEBNext Ultra II RNA Library Prep Kit for Illumina using manufacturers instructions (New England Biolabs, Ipswich, MA, USA). Briefly, mRNAs were initially enriched with Oligod(T) beads. Enriched mRNAs were fragmented for 15 minutes at 94 C. First strand and second strand cDNA were subsequently synthesized. cDNA fragments were end repaired and adenylated at 3ends, and universal adapters were ligated to cDNA fragments, followed by index addition and library enrichment by PCR with limited cycles. The sequencing library was validated on the Agilent TapeStation (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit Fluorometer (ThermoFisher Scientific, Waltham, MA, USA) as well as by quantitative PCR (KAPA Biosystems, Wilmington, MA, USA).