Discovery of Genes Expressed In Basal Endosperm Transfer Cells in Maize Using 454 Transcriptome Sequencing
Total RNA was extracted from the isolated BETCs using the Picopure RNA isolation kit (Arcturus, Part #: KIT0204).  DNase (Qiagen, catalog#: 79254) digestion was performed as directed by the kit. RNA quality was evaluated by using an Agilent (Palo Alto, CA) 2100 Bioanalyzer. We employed the Smart Technology [38] of cDNA construction (Evrogen, Mint-Universal kit, Cat #: SK002; http://www.evrogen.com) to prepare cDNA of BETCs.  Basically, the experiment followed the directions shown in the kit manual. Briefly, the adapter pair CDS-1 (5'-AAGCAGTGGTATCAACGCAGAGTAC(T)30V N-3') (10 uM)  and PlugOligo-1 (5'-AAGCAGTGGTATCAACGCAGAGTACGGGGGP-3') (15 uM) were used for synthesis of cDNA (protocol I , Section VI of the the kit manual). Reverse transcription was carried out on 1 ug total RNA of BETCs. 1 ug total RNA and 1 ul of each CDS-1 and PlugOligo-1 were mixed in 5 ul final volume adjusted by water.  The mix was incubated at 70 degrees C for 2 min and then 42 degrees C for 3 min.  First-strand cDNA synthesis was then initiated by the addition of RT Master Mix (First-Strand Buffer, DTT, dNTP, Mint reverse transcriptase) in a final volume of 10 ul.  The final concentration included: 1X First-Strand Buffer, 2 mM DTT, 1 mM of each dNTP.  The mixed solution was incubated at 42 degrees C for 30 min and again for 1.5 hr after addition of 5 ul IP-solution and then cooled on ice.  1 ul first-strand cDNA was taken to perform ds cDNA synthesis in a final volume of 50 ul, which contains 1X Encyclo PCR Buffer, 0.2 mM of each dNTP, 0.4 uM PCR Primer M1 (5'-AAGCAGTGGTATCAACGCAGAGT-3'), 1X Encyclo Polymerase Mix. We performed 21 cycles with the parameters 95 degrees C for 15 sec, 66 degrees C for 20 sec, and 72 degrees C for 3 min for ds cDNA synthesis by PCR amplification. Amplified cDNA PCR product was purified using QIAquick PCR Purification Kit (QIAGEN, CA), and the final concentration is 55 ng/ul