Drosophila melanogaster
Protocols: Chromatin immunoprecipitation targeting Rad50 were carried out on embryos stage 16 and wandering larval brain. For the embryos, w1118 embryos were collected for 2hrs and incubated at 25°C for 12hrs to reach stage 16. The embryos were dechorionated for 5min using bleach and fixed in 1.8% paraformaldehyde/1xPBS with heptane, under agitation. For the larval brains, 100 w1118 WL3 brains were dissected and fixed in 1.8% paraformaldehyde. Both E16 embryos and WL3 brains were treated as follow The fixation was stopped with 125 mM glycine, the samples were washed three times with PBS, homogenised in RIPA buffer and sonicated. The chromatin was collected, a small aliquot (10% of the total volume) was set aside for the input. The chromatin was incubated with anti-Rad50 antibody and incubated with sepharose -proteinA beads for the immunoprecipitation. The DNA was isolated after decrosslinking by phenol/chloroform extraction. Each ChiP was done in duplicate and yielded around 6ng of DNA for each sample produced with the Rad50 antibody. No DNA was detected in the IgG ChIP controls. The library preparations were performed at the GenomEast platform (IGBMC) using Diagenode MicroPlex Library Preparation kit v3 Instruction Manual. The ChIP samples and inputs were purified using SPRIselect beads (Beckman-Coulter, Villepinte, France) and quantified using the Qubit 4 fluorimeter (Thermo Fischer Scientific, Illkirch, France). ChIP-seq libraries were prepared from 1.8 to 10 ng of double-stranded purified DNA using the MicroPlex Library Preparation kit v3 (C05010001, Diagenode, Seraing, Belgium), according to manufacturer's instructions. In the first step, the DNA was repaired and yielded molecules with blunt ends. In the next step, stem-loop adaptors with blocked 5 prime ends were ligated to the 5' end of the genomic DNA, leaving a nick at the 3' end. The adaptors cannot ligate to each other and do not have single-strand tails, avoiding non-specific background. In the final step, the 3' ends of the genomic DNA were extended to complete library synthesis and Illumina compatible indexes were added through a PCR amplification (7 cycles). Amplified libraries were purified and size-selected using SPRIselect beads (Beckman Coulter) to remove unincorporated primers and other reagents.
strain
w1118
organism
Drosophila melanogaster
organism
Drosophila melanogaster
sex
mixed
scientific_name
Drosophila melanogaster
common name
fruit fly
organism part
whole organism
age
15
developmental stage
embryonic stage 16
genotype
wild type genotype
ENA-LAST-UPDATE
2023-05-23