2.2. RNA extraction and sequencing

Total RNA was isolated from whole leaflets with a method developed specifically for field‐grown soybean (Bilgin, DeLucia, & Clough, 2009), and RNA was treated with the DNA‐free kit (Ambion, Inc., Austin, TX). cDNA libraries were constructed and indexed with the TruSeq RNA Sample Preparation kit, and the average insert size was 361 bp (Illumina, Inc., San Diego, CA). Sixteen samples (four biological replicates at four time points) were sequenced on two randomly assigned lanes with the HiSeq2000 (Illumina, Inc., San Diego, CA). On average, 51.2 million paired‐end 100 nt reads were generated for each sample. We previously reported transcript abundance for 34 soybean aquaporin genes from a separate analysis of these reads (Locke & Ort, 2015).