characteristics: ERC000011
characteristics: ENA-CHECKLIST
characteristics: 2022-10-18T00:27:39Z
characteristics: ENA-FIRST-PUBLIC
characteristics: 2022-10-18T00:27:39Z
characteristics: ENA-LAST-UPDATE
characteristics: SAMEA110410195
characteristics: External Id
characteristics: Institut de Genetique et de Biologie Moleculaire et Cellulaire, Illkirch, France Centre National de la Recherche Scientifique, UMR7104, Illkirch, France Institut National de la Sante et de la Recherche Medicale, U1258, Illkirch, France Universite de Strasbourg, Illkirch, France
characteristics: INSDC center name
characteristics: 2022-10-18T00:27:39Z
characteristics: INSDC first public
characteristics: 2022-10-18T00:27:39Z
characteristics: INSDC last update
characteristics: public
characteristics: INSDC status
characteristics: ERS12508581
characteristics: SRA accession
characteristics: E-MTAB-11966:Sample 1
characteristics: Submitter Id
characteristics: 11 to 14
characteristics: hour
characteristics: age
characteristics: ArrayExpress
characteristics: broker name
characteristics: hemocyte
characteristics: cell type
characteristics: fruit fly
characteristics: common name
characteristics: Protocols: Staged egg laying was carried out to produce E16 embryos as follows. The cross to produce srp(hemo)Gal4/+;UAS-RFP/UAS-gcm-RNAi embryos (with at least 100 females) was transferred to egg laying cages on a yeasted apple juice agar at 25°C. After a pre-lay period of 30 min, the agar plates containing yeast were replaced with fresh plates and flies were left to lay for 3 h at 25°C. Agar plates were then removed and the embryos were raised for 11 h and 40 min at 25°C until they reached embryonic 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 and then the cells were filtered through a 70 µm filter to prepare them for FACS sorting. The cells were sorted using FACS Aria II (BD Biosciences) at 4°C in three independent biological replicates. Live cells were first selected based on the forward scatter and side scatter and only single cells were taken into account. OregonR cells were used as a negative control to set the gate for the sorting of RFP positive cells only. RFP positive hemocytes were collected in 1 ml of TRI reagent (MRC) for RNA extraction. For the wandering L3 srp(hemo)Gal4/hmlΔRFP;UAS-gcm-RNAi/+ hemocytes, staged lay of 3 h were carried out at 25°C to prevent overcrowding of the vials (between 50 and 100 embryos per vial) and wandering larvae were collected 108-117 h AEL, bled in cold PBS containing PTU (Sigma-Aldrich P7629) to prevent hemocyte melanization (Lerner and Fitzpatrick, 1950), filtered through a 70 µm filter to isolate them and sorted by FACS as described for the embryonic hemocytes. The purity of the sorted populations was assessed prior to the collection of the sample for RNA extraction by carrying out a post-sort step. The FACS sorter was set up to produce hemocyte pools displaying at least 80% of purity on the post-sort analysis. At least 50,000 sorted cells were homogenized then left at room temperature (RT) for 5 min to ensure complete dissociation of nucleoprotein complexes. 0.2 ml of chloroform was added to each sample followed by centrifugation at 12,000g for 15 min at 4°C. The upper aqueous phase containing the RNA was collected and transferred to a fresh autoclaved tube. 0.5 ml of 2-propanol were added, and the samples were incubated for 5-10 min at RT. The RNA was precipitated by centrifugation then washed with 1 ml of 75% ethanol then precipitated again and air dried. 20 µl of RNase-free water was added to each sample before incubation at 55°C for 15 min. Single-end mRNA-Seq libraries were prepared using the SMARTer (Takara) Low input RNA kit for Illumina sequencing.
characteristics: description
characteristics: embryonic stage 16
characteristics: developmental stage
characteristics: srp(hemo)Gal4/+;UAS-RFP/UAS-gcm-RNAi
characteristics: genotype
characteristics: RFP-positive
characteristics: immunophenotype
characteristics: pool of embryos
characteristics: individual
characteristics: Drosophila melanogaster
characteristics: organism
characteristics: Drosophila melanogaster
characteristics: organism
characteristics: whole organism
characteristics: organism part
characteristics: Drosophila melanogaster
characteristics: scientific_name
characteristics: mixed
characteristics: sex
characteristics: srp(hemo)Gal4/+;UAS-RFP/UAS-gcm-RNAi
characteristics: strain
characteristics: Sample 1
characteristics: title