F1EmbryoNonNormalized
RNA extraction:   A single embryo approximately 8.5days old was harvested and stored in RNAlater (Ambion, AM7020). RNA was extracted using TRIzol Plus RNA Purification Kit (Invitrogen cat no: 12183-555) including an on column DNase digestion using PureLink DNase (Invitrogen 12185-010).  The RNA was quantitated using nanodrop then diluted 1:250 and 1:125 in TE and quality checked via use of an Agilent Pico Chip (5067-1513).    CDNA synthesis:   The cDNA samples were prepared using a modification of the protocol developed by Galina Aglyamova, Eli Meyer, Mikhail V. Matz, Jade Buchanan-Carter, Keithanne Mockaitis, and John K. Colbourne using the SMART PCR cDNA Synthesis Kit (Clontech, cat no: 634902).  For the first strand cDNA synthesis,  a single 5uL reaction consisting of 300 ng RNA, 1uL of 12uM Cap-Trsa-CV primer (AAG CAG TGG TAT CAA CGC AGA GTC GCA GTC GGT ACT TTT TTC TTT TTTV), and 1uL of 12uM SMART II A Oligonucleotide were incubated at 72 degrees C for 2 minutes, followed by a 2 minute incubation on ice. To the reaction, 5uL of 2uL 5X First-Strand Buffer, 1uL 20mM DTT, 1uL of 10uM each dNTP mix, and 1uL Invitrogen Superscript Reverse Transcriptase (Invitrogen, cat no: 18064-014 )were added and incubated at 42 degrees C for 1 hour.  The reaction was terminated by placing on ice. Five 100uL amplification reactions were carried out using 2uL of the above single stranded cDNA.  Each reaction consisted of 80uL water, 10uL 10X Advantage 2 PCR Buffer, 2uL 50XdNTP Mix, 4uL 12uM 5'PCR Primer IIA, 2uL 50X Advantage 2 Polymerase Mix (Clontech, cat no: 639201), and 2uL template.  The reactions were incubated at 95 degrees C for 1 minute followed by 20 cycles of 95 degrees C for 7 seconds, and 68 degrees C for 6 minutes (Hale, M.C., 2009).  The product was then purified using QIAGEN QIAquick PCR Purification Kit, and visualized using Agilent DNA 7500 Chip.  A sample of at least 5ug of cDNA in 120-140uL TE was then handed to the Emory Genomics Core for cDNA Library Preparation and sequencing