RNA-seq of Embryo of maize B73
Total RNA (10 μg) from each sample was extracted using RNeasy Mini Kit (Qiagen), according to the manufacturer’s protocol. mRNA was isolated from total RNA using 7 μl of oligo dT on Sera-magnetic beads and 50 μl of binding buffer. mRNA was fragmented by metal hydrolysis in RNA fragment buffer (Ambion) for 2 min at 70C. The reaction was stopped by adding 2 μl of fragmentation stop solution (Ambion). The fragmented RNA was converted to double-stranded cDNA. After polishing the ends of the cDNA, an adenine base was added at the 3’ ends, after which Illumina multiplex adaptors were ligated. The ligated DNA was separated on 2% agarose gel and 300-nt targeted DNA was extracted. DNA was purified from the gel using the Qiagen Gel extraction kit. The purified DNA was amplified by 15 cycles of PCR, and the PCR DNA was then purified on the Qiagen PCR purification kit to obtain the final seq library for sequencing. The DNA concentration of the seq library was determined on Qubit (Invitrogen).