Strand-specific RNA-seq primer design

Genome sequences from the MIPS U. maydis database (MUMDB; [57 (link)]) were used to design strand-specific first-strand synthesis primers, as well as PCR primers, as described in [15 (link), 58 (link)]. All primers used in this study are listed in Additional file 21. Antisense strand-specific first-strand synthesis primers were designed based on RNA-seq-predicted transcript structures. First-strand synthesis primers included oligo(dT)₁₆, DEPC-treated H₂O, a sense-specific first-strand primer or an antisense-specific first-strand primer. First strand synthesis was conducted on 200 ng of DNase I-treated RNA, using the TaqMan Gold RT-PCR kit (Applied Biosystems) following methods outlined in [58 (link)]. cDNA was diluted eightfold (1:7) with dH₂O. PCRs were conducted using the AmpliTaq Gold DNA Polymerase Kit (Applied Biosystems) following the manufacturer’s recommended protocol. One third of the PCR products were visualized using agarose gel electrophoresis (1X TAE) and were subsequently stained with 0.3 μg/mL ethidium bromide (BioShop).