GSM8070114: Nema-JN2; Pinus densiflora; RNA-Seq
One gram of pinewood nematode-infected needles was ground in liquid nitrogen and mixed with 15 ml of extraction buffer containing 100 mM Tris-HCl (pH 8), 2% CTAB, 30 mM EDTA, 2 M NaCl, 0.05% spermidine, 2% polyvinylpolypyrrolidinone, 2% 2-mercaptoethanol, and 1.5 mg/ml proteinase K. The suspension was incubated at 42 °C for 90 min and then extracted with 15 ml chloroform-isoamyl alcohol (24:1), with total RNAs precipitated using 10 M LiCl. After washing with 70% ethanol, the RNA pellets were dissolved in DEPC water. Using the TruSeq RNA Sample Preparation Kit (Illumina, CA, USA), 1 µg of total RNAs was processed to produce cDNA libraries. These libraries were sequenced on a HiSeq 4000 platform (Illumina) by Macrogen (Seoul, Korea), generating 101-bp paired-end reads.