Illumina HiSeq 2000 sequencing; RNA-seq of FACS purified cardiomyocytes from Drosophila melanogaster embryos at stage 13-16.
RNA-seq of FACS purified cardiomyocytes from Drosophila melanogaster embryos at stage 13-16.
Embryos were collected from 4 large cylindrical population cages, each seeded with ~70 g of newly eclosed flies (representing ~70000 flies). After performing three 1 h pre-lays, 3 h embryo collections using 3 standard molasses plates (3g agar, 3g sugar, 30mL apple juice, 100mL water, 1mL propionic acid) streaked with yeast paste) by cages typically yielded 3-5 g dry weight of tightly staged embryos. Cages were maintained at 25C and 60% of hygrometry. Embryos were washed and transfer into large sieves. After dechorionation (2.5 min in 400 ml 3% NaOCl (50% bleach)) embryos were transferred to a Nitex membrane (5x5 cm) to be dried with a paper towel. Then they were placed into a 50 ml Falcon tube with 9.5ml Cross-linking solution (50 mM Hepes, 1 mM EDTA, 0.5 mM EGTA, 100 mM NaCl, pH 8), 485 microl 37% Formaldehyde and 30ml n-Heptane and shaked vigorously at room temperature (20-25C) for 15 min. They were washed once with 30 ml PBS/Glycin/Triton (125 mM glycine, 0.1% Triton in PBS), once with 50 ml ice-cold PBT and suspended in 10 ml PBT (PBS, 0.1% Triton). After drying, cross-linked embryos were freezed in liquid nitrogen and stored at -80C. Cells were mechanically and enzymatically dissociated and sorted using a FACS Aria II (Becton Dickinson) equipped with a 488 nm laser. 50 mg of staged embryos typically retrieved 2 x 104 sorted (cardiac) cells and 50ng of total RNA (Trizol extracted). Then use Oligo-dT beads to extract mRNA. Total RNA (50 ng for each biological replicate) were amplified with Amino Allyl MessageAmp II kit (Applied Biosystems AM1753) and resulting amplified mRNA were used for library preparation.
Experimental Factor: 2
replicate