Sequencing of cDNA from heart tissue of pooled Embryo
RNA extraction:   Three embryos, approximately 12.5days old, were 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 in TE and quality checked via use of an Agilent Pico Chip (5067-1513).    CDNA synthesis:   The cDNA samples for each embryo were prepared separately 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, For each embryo a single 5uL reaction consisting of 1ug RNA, 1uL of 12uM Cap-Trsa-CV primer (AAG CAG TGG TAT CAA CGC AGA GTC GCA GTC GGT ACT TTT TTC TTT TTTV), 1uL of 12uM SMART II A Oligonucleotide, and water, were incubated at 72 degrees C for 2 minutes, followed by a 2 minute incubation on ice. To each 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, per embryo sample, 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 15 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.  The embryo samples were pooled using 1.7 ug cDNA per sample in 120-140uL TE and the single pooled sample was then handed to the Emory Genomics Core for cDNA Library Preparation and sequencing.