title: GSM8196077: VAL_ab_rep3; Citrus reticulata x Citrus trifoliata; RNA-Seq
library_construction_protocol: The tissue was ground in liquid nitrogen using mortar and pestle and stored at -80 °C until use. Approximately 100 mg of ground tissue were used to extract RNA using the Qiagen RNeasy plant mini kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. RNA quality and concentration were measured using the QUBIT RNA fluorometry method (Invitrogen, Waltham, MA) and an Agilent Bioanalyzer (Agilent, Santa Clara, CA). An amount of 250 ng of high-quality total RNA with an RNA integrity number (RIN) of 7 or higher was used for the library construction using the reagents provided in the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, Ipswich, MA) and the NEBNext Ultra II Directional RNA Library Prep Kit (New England Biolabs) according to the manufacturer's instructions. Briefly, 100 ng of total RNA were used for mRNA isolation using the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs). The poly A enriched RNA was fragmented in NEBNext First Strand Synthesis Buffer. Synthesis of double stranded cDNA was done using the 2nd strand master mix provided in the kit, followed by end-repair and dA-tailing.  At this point, Illumina adaptors were ligated to the sample.  Finally, the library was amplified, and followed by purification with AMPure beads. Thirty-nine barcoded libraries were pooled equimolarly for sequencing simultaneously for 0.52 lanes of NavaSeq 6000 S4 2x150 cycles