PI-TPC-GEO
The maize pericarp transcriptome
The preparation of mRNA-Seq libraries was performed according to instructions from the Illumina mRNA-Seq Sample Preparation Kit (RS-100-0801 Illumina). Total RNA extracted from P1-rr and P1-ww pericarps harvested at 14 and 25 DAP and silks harvested 1-2 days post emergence were treated with DNaseI (Promega) twice to ensure no genomic DNA contamination. Two µg of total RNA was subjected to two rounds of hybridization to oligo(dT) beads (Illumina). The polyA-enriched mRNA was fragmented by heating at 94 °C for 5 minutes in Fragmentation Buffer supplied by Illumina, followed by ethanol precipitation. The fragmented mRNA was added into 1x First Strand Buffer (2.5mM dNTP, 10mM DTT, RNaseOUT, and random primers mix). After adding Superscript II, first strand cDNA synthesis was performed at 25°C for 10 minutes, 42°C for 50 minutes and 70°C for 15 minutes. The second strand cDNA synthesis was carried out in GEX Second Strand Buffer (25mM dNTP, 1µl RNase H and µl DNA Pol I for 2.5 hours at 16°C), and purified by PCR Purification Kit (Qiagen). After end repair and A-base addition, adapter ligation was performed using custom made adapters or Illumina multiplexing system. For custom-made adapters, reverse oligo-adapters consisting of SLX-R_P1 and SLX-R_P2 and forward oligo adapters consisting of SLX-N_P1 and SLX-N_P2, where underlined nucleotides (see Table S6) corresponds to a barcode sequence, which was either AATCG, AACAC, AACGT, or AACTG in P1-ww at 14 or 25 DAP and P1-rr 14 or 25 DAP, respectively. For using Illumina multiplexing system, the overall procedure was according to the manufacturer’s instructions (MultiplexPE_SamplePrep_1005361_RevB). We used Index 1 (ATCACG) or 2 (CGATGT) for P1-rr or P1-ww silk, respectively. For both procedures, ligated fragments were subjected to electrophoresis for selection of fragments of ~300 bp in length, followed by PCR amplification (20 cycles). PCR products were purified with Agencourt AMPure (Beckman Coulter). Library DNA was checked for concentration and size distribution in an Agilent Bioanalyzer before being subjected to Illuimina GA sequencing using the OSUCCC Nucleic Acid Shared Resource-Illumina GAII Core and the OARDC Molecular and Cellular Imaging Center (The Ohio State University, Ohio).
P1 encodes an R2R3-MYB transcription factor responsible for the accumulation of insecticidal flavones in maize silks and red phlobaphene pigments in pericarps and other floral tissues, which contributed to making P1 an important visual marker since the dawn of modern genetics. We conducted RNA-Seq usin pericarps at two different stages, 14 and 25 days after pollination (DAP). High-throughput sequencing using the Illumina platform resulted in the generation of ~20 million high quality reads, from which ~90% aligned to the recently completed maize genome sequence corresponding to ~5 million reads for each one of the four samples.