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 collection 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, 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 by the Chromium Controller during the encapsulation.  From the remaining 30µl of cDNA generated during the scRNA-seq library preparation, 300 ng were used for targeted PCR amplification. The region containing the specific enhancer barcode and the 10X Genomics barcodes (cellular barcode and UMI) was amplified with the Q5 Hot Start High Fidelity polymerase (New England Biolabs). The forward primer binds to the 19nt sequence common to all constructs (GACGTCATCGTCCTGCAGG) and the reverse primer to the TruSeqRead1 sequence added during the scRNA-seq library preparation (CTACACGACGCTCTTCCGATC). The cycling conditions were as follows:  Initial denaturation at 98°C for 10 seconds; then 25 cycles at 98°C for 10 seconds, 68°C for 30 seconds, 72°C for 20 seconds; then an elongation step at 72°C for 5 min. The PCR product was purified using SPRIselect beads (Beckman Coulter) and 100 ng used to generate the final libraries using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs). The libraries were indexed for multiplexing using NEBNext multiplex oligos kit for Illumina (New England Biolabs)
strain
nos-phiC31; attP40
replicate
2
individual
mix pool of embryos
organism
Drosophila melanogaster
organism
Drosophila melanogaster
collection date
not collected
sex
mixed
scientific_name
Drosophila melanogaster
common name
fruit fly
organism part
embryo
developmental stage
embryonic stage 6
genotype
nos-phiC31; attP40
isolate
not applicable
age
2.5 to 3.5
geographic location (country and/or sea)
not collected