Nebnext ploy a mrna magnetic isolation module

Tree tissue samples were ground in liquid nitrogen using a bead beater and Lysing Matrix A (MP Biomedicals LLC, Solon, OH). Approximately 50 mg of each ground sample was used for RNA extraction using the method of Chang et al. [115 ]. The RNA was further treated with DNAase and concentrated using RNA Clean and Concentrator-5 (Zymo Research). RNA samples were submitted to GENEWIZ for library preparation and RNA sequencing (Illumina HiSeq3000, 2 × 150 bp, with PolyA Selection). For in vitro-grown fungal tissue, approximately 10 mg of the freeze-dried fungal tissue for each sample was homogenized using a bead beater. Total RNA was extracted using the RNeasy Plant Mini kit according to the manufacturer’s instruction (QIAGEN). The Interdisciplinary Center for Biotechnology Research (ICBR) NextGen DNA Sequencing core, University of Florida (UF) performed mRNA isolation using NEBNext Ploy(A) mRNA Magnetic Isolation module (New England Biolabs, catalog # E7490) and RNA library construction with NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs, catalog # E7530) according to the manufacturer’s user guide. Paired-end, 2 × 100 cycle sequencing was performed at the ICBR on two lanes of the Illumina HiSeq3000 instrument using the clustering and sequencing reagents provided by Illumina (San Diego, CA, USA).

The RNA-sequencing libraries were constructed at ICBR Gene Expression and Genotyping using NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina (NEB, Ipswich, MA, USA), following manufacturer’s recommendations. Basically, 500 ng of high-quality total RNA (RIN ≥ 7) was used for mRNA isolation using NEBNext Ploy(A) mRNA Magnetic Isolation module (New England Biolabs, Ipswich, MA, USA). This was then followed by RNA library construction with NEBNext Ultra II Directional Lib Prep (New England Biolabs, Ipswich, MA, USA), according to the manufacturer’s user guide. Briefly, RNA is fragmented in NEBNext First Strand Synthesis Buffer by heating at 94 °C for the desired time. This step is followed by first strand cDNA synthesis using reverse transcriptase and oligo dT primers. Synthesis of ds cDNA is performed using the 2nd strand master mix provided in the kit, followed by end-repair and adaptor ligation. Finally, the library is enriched (each library has a unique barcode) by 11 cycles of amplification, and purified by Agencourt AMPure beads (Beckman Coulter, Atlanta, GA, USA, catalog # A63881). Finally, each 14 individual libraries were pooled (total of 3 pools) with equimolar and sequenced by Illumina HiSeq 3000 2X 100 cycles run for total of 1 run (Illumina Inc., San Diego, CA, USA).