GSM7295029: IAA,Day15,Rep2; Picea abies; RNA-Seq
Total RNA was extracted using a modified CTAB/LiCl method(Azevedo et al., 2003). Briefly, frozen tissue was ground into a fine powder. Extraction buffer was prepared (100 mM Tris-HC1 (pH 8), 2% (w/v) CTAB, 30 mM EDTA, 2 M NaCI, 0.05% (w/v) spermidine, 2% (w/v) PVPP, 2% (v/v) 2-mercaptoethanol, proteinase K (10 mg/mL) to a final concentration of 1.5 mg/mL) and warmed for 10 min at 42℃, then added to the ground frozen tissue and incubated for at 42℃for 90 min.  Chloroform-isoamyl alcohol (24:1 [v/v]) was added to extract RNA. After vortexing and centrifuging at 15,000 g for 15 min at 4℃, the top aqueous phase was transferred to a new tube and ¼ vol of 10 M LiC1 was added, allowing overnight precipitation at 4℃. Samples were centrifuged at 15,000 g for 30 min and supernatant discarded. Finally, the pellet was washed with 2 M LiCl twice and dissolved in RNase-free water. RNA concentration was measured using Qubit 2.0. The RNA Integrity Number (RIN) was analyzed by using an Agilent 2100 Bioanalyzer with RNA 6000 Nano kit. The RINs of all samples were above 8.0. mRNA library preparation (poly A enrichment) by Novogene