Genome-wide Transcriptome and Proteome Profiling Indicate an Active Role of Alternative Splicing During De-etiolation of Maize Seedlings
Total RNA was extracted with the RNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA) according to the standard protocol, and mRNA was purified from total RNA using the Dynabeads mRNA Purification Kit (Invitrogen, USA). The polyA containing mRNA was purified with oligo-dT attached magnetic beads and RNA was fragmented and primed for cDNA synthesis during elution of the polyA RNA according to the standard protocol. The first stranded cDNA was synthesized using SuperScript II Reverse Transcriptase (Invitrogen, USA) and random primers (Invitrogen, USA), followed by second strand cDNA synthesis. The overhangs resulting from fragmentation were converted into blunt ends using an End Repair Mix (Invitrogen, USA). The 3' to 5' exonuclease activity of this mix removes the 3' overhangs and the polymerase activity fills in the 5' overhangs. After adenylation of 3 ends of DNA fragments, the Adaptors provided by Illumina were ligated. The fragments were amplified by PCR and 400-500 bp cDNA fragments were purified and sequenced with Illumina system Hiseq2000. Sequencing was performed by Beijing BerryGenomics Co., Ltd. (China)