GSM6268282: NejireKD_stage5_rep3; Drosophila melanogaster; RNA-Seq
For each biological replicate, 20 hand-sorted stage 2 or stage 5 embryos  were collected in 50μl of TRIzol™ (Thermo Fisher Scientific 15596026), homogenized with pestle and stored at -20°C. For nuclear RNAseq, nuclei pellets obtained from 300 stage 5 embryos were similarly collected and homogenized in 50μl of TRIzol™. RNA was then extracted in 500μl TRIzol mixed vigorously for 15 seconds with 100μl chloroform. For nuclear RNA-seq only, 1μl of 1:1000 diluted ERCC RNA spike-in (Ambion™ 4456740) was added with the remaining 450μl TRIzol before chloroform extraction, in order to normalize for a potential global variation of RNA quantity/nucleus upon knockdown. Samples were centrifuged at 12000g 4°C for 15min and the aqueous phase was mixed again with an equal volume of 300μl chloroform and centrifuged again at 12000g 4°C for 15min. The aqueous phase was mixed with 300μl of isopropanol and 20μg of glycogen and total RNA was let precipitate over night at -20°C. The day after, samples were centrifuged at 12000g 4°C for 30min and the RNA pellet was washed twice with 80% EtOH. Air dried RNA pellets were resuspended in 20μl of TURBO™ DNase solution (Invitrogen™ AM2238) and incubated for 30min at 37°C, according to manufacturer's instructions. EDTA was added to a final concentration of 15mM and DNase was heat inactivated at 75°C for 10min. Libraries were prepared by amplification of tagmented DNA fragments using NEBNext® Q5® HiFi PCR Master Mix (NEB, Cat. M0543). Primer sequences were taken from Buenrostro et al., 2015. Libraries were quality controlled by capillary electrophoresis on the Fragment Analyzer system (Advanced Analytical).