Instrument: Illumina HiSeq 2500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: mRNA libraries: Total RNA was extracted using the CTAB method and purified by phenol/chloroform extraction. mRNA libraries: Fifteen to twenty micrograms of total RNA was processed in preparation for Illumina sequencing, according to the previous report (D. Burkart-Waco et al., Plant Cell 25, 2037-2055 (2013)). Briefly, mRNA was purified using the Dynabeads mRNA purification kit (Life Technologies). Next, cDNA was synthesized via random priming using Superscript III (Life Technologies), followed by heat inactivation for 5 min at 65°C. Second-strand cDNA was synthesized using the second-strand buffer (200 mM Tris-HCl, pH 7.0, 22 mM MgCl2, and 425 mM KCl), DNA polymerase I (NEB) and RNaseH (NEB) with incubation at 16°C for 2.5 h. Double-stranded cDNA was purified using AMPure with a 1.8 : 1 (v/v) AMPure to reaction volume ratio. The resulting double-stranded cDNAs were subjected to fragmentation and following library construction, as follows. Approximately 1.0 μg of cDNA was fragmented using NEBNext dsDNA Fragmentase (New England BioLabs; NEB) for 40-60 min at 37°C and cleaned using Agencourt AMPure XP (Beckman Coulter Genomics) for size-selection. To select fragments ranging between 200 and 600 bp, 25 μl AMPure were added to the initial 50 μl reaction. After a brief incubation, 72 μl of the supernatant was transferred to a new tube, and an additional 12 μl water and 36 μl AMPure were added. After a second brief incubation, the supernatant was discarded and the DNA was eluted from the beads in 20 μl of EB, as recommended. Next, DNA fragments were subjected to end repair using NEB’s End Repair Module Enzyme Mix, and A-base overhangs were added with Klenow (NEB), as recommended by the manufacturer. End repair and A-base addition were both followed by AMPure cleanup using 1.8 : 1 (v/v) AMPure / reaction. Barcoded NEXTflex adaptors (Bioo Scientific) were ligated at room temperature using NEB Quick Ligase (NEB), following the manufacturer’s recommendations. To remove contamination of self-ligated adapter dimers, libraries were size-selected using AMPure in 0.8 : 1 (v/v) AMPure : reaction volume, in order to select for adapter-ligated DNA fragments at least 300-bp long. Half of the eluted DNA was enriched by PCR reaction using Phusion 2X HF master mix (NEB), with the following PCR conditions: 30 s at 95°C; 10 cycles of 10 s at 95°C, 30 s at 65°C, and 30 s at 72°C and a final extension step of 1 min at 72°C. Enriched libraries were purified with AMPure (0.8 : 1 v/v AMPure to reaction), and quality and quantity were assessed using the Agilent BioAnalyzer (Agilent Technologies) and Qubit fluorometer (Invitrogen). The constructed libraries were sequenced on Illumina’s HiSeq 2500 sequencer (150-bp paired-end reads).