RNA sequencing of wheat Ms2 mutant among different tissues

Ms2 isogenic lines, LM15 and LM15RMs2, were choosed for RNA sequencing. Plants were grown in the greenhouse with a 16h photoperiod and a daytime and nighttime temperature of 22°C and 15°C, respectively. At the S2 stage, when an auricle distance between the penultimate and flag leaf was between 3 to 5 cm, we collected four types of samples: fertile anthers (FA) of LM15, sterile anthers (SA) of LM15RMs2, pistils (P) of LM15 and LM15RMs2, and flag leaves (L) of LM15 and LM15RMs2. Tissues were stored in liquid nitrogen during collection and then at -80 °C for long-term storage. Three biological replicates were made for each type of tissue. Total RNA was isolated using TRIzol (Invitrogen) and RNA integrity was evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). The Berry Genomics Company prepared the sequencing libraries (ca. 500bp per insert) and performed the high-throughput sequencing (125bp PE reads) on HiSeq2500.