Drosophila melanogaster
Protocols: Freshly hatched adults from the pool of transgenic flies containing our library of candidate enhancers were combined in 5 embryo collection vials with standard apple cap plates. After three 45-minute pre-lays, Drosophila embryos were collected on apple juice plates for one hour and incubated for another 2.5 hours at 25°C. We verified that the collected embryos mostly span developmental stages 5 to 7. The embryos were dechorionated using 2,6% bleach for 2 minutes, washed with water and PBS + 0.1% Triton X-100, and finally resuspended in 1 ml of ice-cold PBS + 0.1% Triton X-100 and kept on ice. Embryos were washed with ice-cold PBS, resuspended in 10 mL of dissociation buffer (PBS 1X + 0.1% BSA), and dissociated on ice in a Dounce homogenizer with gentle strokes of the loose pestle. This process was repeated for all collected embryos, using a small number of embryos each time. The cell suspension was transferred to 15 mL Falcon tubes and centrifuged for 10 minutes at 40g at 4°C to pellet debris. Cells in the supernatant were transferred to new Falcon tubes and centrifuged again for 10 minutes at 800g at 4°C. The supernatant was discarded, and cell pellets were combined and resuspended in 0.5 mL of dissociation buffer. Two additional rounds of centrifugations, each for 5 minutes at 800g at 4°C were performed to remove as much debris as possible. Cells were counted using a Malassez counting chamber and the concentration was adjusted to 900 cells/µl in the dissociation buffer. Single cells were encapsulated into droplets in the Chromium Controller instrument for cell lysis and barcoded reverse transcription of mRNA. 40 µl of cDNA were recovered, and 10 µl (25%) were used for amplification, fragmentation, and Illumina library construction.  Cells were encapsulated with the 10X chromium device. RNA extraction was performed following the10XGenomics Chromium Next GEM Single Cell 3ʹ Kit v3.1 Library preparation was performed with the 10X library kit provided with the Chromium Next GEM Single Cell 3ʹ Kit v3.1
ENA first public
2024-01-01
INSDC center name
Institut de Génomique Fonctionnelle de Lyon
INSDC status
public
Submitter Id
E-MTAB-14447:Sample 2
age
2.5 to 3.5
broker name
ArrayExpress
cell_type
whole organism
collection date
not collected
common name
fruit fly
developmental stage
embryonic stage 6
genotype
nos-phiC31; attP40
geographic location (country and/or sea)
not collected
individual
mix pool of embryos
isolate
not applicable
organism part
embryo
replicate
2
sample name
E-MTAB-14447:Sample 2
scientific_name
Drosophila melanogaster
sex
mixed
strain
nos-phiC31; attP40