1000-01-01
ENA first public
ERC000011
ENA-CHECKLIST
Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France Centre National de la Recherche Scientifique, UMR7104, Illkirch, France Institut National de la Santé et de la Recherche Médicale, U1258, Illkirch, France Université de Strasbourg, Illkirch, France
INSDC center name
public
INSDC status
ERS20948757
SRA accession
E-MTAB-14413:E16 glia replicate 3
Submitter Id
embryonic stage 16
age
ArrayExpress
broker name
glial cell
cell_type
not collected
collection date
fruit fly
common name
Protocols: Embryonic cells were isolated as follows. Staged egg laying was carried out to produce E16 embryos. repo-nRFP, elav-nRFP and Oregon-R strains were amplified, staged lays were done on yeast apple juice agar at 25°C. After a pre-lay period of 30 minutes, the agar plates and yeast were replaced with fresh plates and flies were left to lay for 3 hours at 25°C. Agar plates were removed and kept at 25°C, embryos were collected 11 hours and 40 minutes AEL, when they had reached stage 16. Embryos were then isolated from the medium and washed on a 100 μm mesh. The collected embryos were transferred into a cold solution of phosphate-buffered saline (PBS) in a Dounce homogenizer on ice. The embryos were dissociated using the large clearance pestle then the small clearance pestle, then filtered (70 μm filter). Cells were sorted using FACS Aria II (BD Biosciences) at 4°C in three independent biological replicates for each genotype. Live cells were first selected based on the forward scatter and side scatter and only single cells were sorted according to the RFP signal. Oregon-R cells were used as a negative control to set the gate and sort the RFP positive cells (Sup. Figure 3-n), which were collected directly in TRI reagent (MRC) for RNA extraction. Around 100 000 cells were sorted for each replicate. The purity of the sorted populations was assessed by carrying out a post-sort step. The FACS sorter was set up to produce cell pools displaying at least 80% of purity on the post-sort analysis. L3 neurons and glia were purified from elav-nRFP and repo-nRFP and  larvae, respectively. Staged lays of 3 hours were carried out at 25°C and wandering larvae were collected 108–117 hours AEL. For neurons and glia isolation, larval brains were dissected in cold PBS on ice and transferred to tubes containing 0.5 μg of collagenase IV (Gibco, Invitrogen) in 220 μL of PBS. Brains were incubated at 37°C on a Thermomixer with shaking at 500 rpm for 20 minutes. Then the brains were dissociated by pipetting up and down with 10-gauge needles and syringes and filtered through a 70 µm filter. Cells were sorted using FACS Aria II (BD Biosciences) at 4°C in three independent biological replicates for each genotype. Live cells were first selected based on the forward scatter and side scatter and only single cells were sorted according to the RFP signal. Oregon-R cells were used as a negative control to set the gate and sort the RFP positive cells (Sup. Figure 3-n), which were collected directly in TRI reagent (MRC) for RNA extraction. Around 100 000 cells were sorted for each replicate. The purity of the sorted populations was assessed by carrying out a post-sort step. The FACS sorter was set up to produce cell pools displaying at least 80% of purity on the post-sort analysis.  Sorted cells were homogenized in TRI reagent (MRC) for 5 minutes at room temperature (RT) to ensure complete dissociation of nucleoprotein complexes. 0.2 mL of chloroform was added to each sample followed by centrifugation at 12,000 g for 15 minutes at 4°C. The upper aqueous phase containing the RNA was collected and transferred to a new tube. 0.5 mL of 2-propanol were added, and the samples were incubated for 10 minutes at RT. The samples were then centrifuged at 12,000 g for 10 minutes to precipitate the RNA. The RNA pellet was then washed with 1 mL of 75% ethanol then precipitated again at 7,500 g for 5 minutes and air dried. 20 µL of RNase-free water was added to each sample before incubation at 55°C for 15 minutes.  Single-end polyA+ RNA-Seq (mRNA-seq) libraries were prepared using Clontech SMART-Seq v4 Low Input RNA Kit.
description
embryonic stage 16
developmental stage
repo-nRFP
genotype
not collected
geographic location (country and/or sea)
not applicable
isolate
Drosophila melanogaster
organism
Drosophila melanogaster
organism
embryonic glia
organism part
Drosophila melanogaster
scientific_name
male and female
sex
repo-nRFP
strain
E16 glia replicate 3
title