Ovules from thirty cleared florets were examined for each group. If the cleared sample showed AIs in less buy inhibitor than 30% of the Inhibitors,Modulators,Libraries ovaries and the remaining ovaries were at an earlier developmental stage, then florets stored in RNALater solution from the same section of inflorescence were used for ovule dissection. About 40 ovules per sample were collected and total RNA was extracted Inhibitors,Modulators,Libraries from the ovules with RNAqueous Micro Kit. RNA integrity and quantity were analyzed with an Agilent 2100 Bioanalyser at the Interdisciplinary Center for Biotechnology Research of the University of Florida. RNA amplification and ds cDNA synthesis for Roche 454 sequencing With total RNA as starting material, mRNA was ampli fied by T7 based in vitro transcription following the manual of TargetAmp 2 Round aRNA Amplification Kit 2.
0. Size range and quan tity of the amplified mRNA were measured by both gel electrophoresis and Agilent 2100 Bioanalyser analysis. For each Inhibitors,Modulators,Libraries sample, an equal amount of amplified mRNA from the three biological replicates was pooled for ds cDNA synthesis following the protocol developed by the Schnable lab. Size range and quantity of ds cDNA were also analyzed by both gel electrophoresis and using the Agilent 2100 Bioanalyser before submitting the sam ples for sequencing. 454 sequencing and processing About 6 ug of ds cDNA from both PS26 and BC8 was submitted to the Genome Sequencing Center at Washington University for 454 FLX sequencing. Sam ples of cDNA were subjected to mechanical shearing, size selected, and blunt end fragments were ligated to short adaptors, which provided primer target sites for both amplification and sequencing.
Sequencing files were sub mitted to the Sequence Read Archive at NCBI view studies. The Multifunctional Inertial Reference Assembly program was used to Inhibitors,Modulators,Libraries process and assemble the sequences from each library. Adaptor sequences and low quality sequence reads were removed prior to assembly. The assembly Inhibitors,Modulators,Libraries was run as a de novo, 454 EST project with accurate assembly and polyA T clipping. Each library of contig assemblies from PS26 and BC8 was converted to a database and analyzed with the BlastN program provided by the RCC at the University of Georgia. The PS26 library contigs were chosen as queries and the BC8 library was chosen as the database. The BlastN analysis was performed with an E value cut off of e 100.
The BlastN output was parsed using an ntity over at least 100 bp were selected for further analysis. BLAST analysis of the selected contigs BlastX was used to analyze sequences mapping to the ASGR carrier chromosome by searching against the NCBI databases. directly A BlastN ana lysis was conducted on contigs without significant BlastX hits to search for similar ESTs from other species. The most significant EST hit with an e value of at least e 20 was used for BlastX query to search for putative encoding proteins.