Drosophila melanogaster
Protocols: 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.
ENA first public
2017-01-17
ENA last update
2017-01-17
External Id
SAMEA50755168
INSDC center alias
EMBL
INSDC center name
European Molecular Biology Laboratory
INSDC first public
2017-01-17T17:02:28Z
INSDC last update
2017-01-17T09:36:25Z
INSDC status
public
Submitter Id
E-MTAB-4763_2:Tin-GFP_R2
broker name
ArrayExpress
common name
fruit fly
developmental stage
stage13-16
genotype
wild type genotype
replicate
2
sample name
E-MTAB-4763_2:Tin-GFP_R2
sex
mixed sex
strain
Tin-GFP