title: GSM9326789: Ctrl DSS 1; Drosophila melanogaster; RNA-Seq
library_construction_protocol: QIAzol lysis buffer, following total RNA isolation using the miRNeasy Micro Kit (Qiagen). Libraries were prepared using the QuantSeq 3'mRNA-Seq Library Prep Kit (Lexogen). Briefly, up to 150 ng of RNA were used for first strand synthesis, followed by RNA template removal and second strand synthesis initiated by random primers containing compatible linker sequences at their 5' end. In-line barcodes were introduced during second strand synthesis, followed by magnetic bead-based purification. The resulting libraries were amplified for up to 19 cycles and purified. Library quantities were assessed using a Qubit 4 Fluorometer and the Qubit dsDNA HS Assay Kit (Invitrogen) and quality was assessed using the Agilent Tapestation 4150 and the High Sensitivity D1000 assay kit, according to manufacturers' instructions. The quantified libraries were pooled equimolarly, and processed for Adapter Conversion PCR Amplification using the Universal Library Conversion Kit (App-A) (MGI Tech Co., Ltd.) according to manufacturer's instructions. The quantity of the purified adapter conversion PCR (AC-PCR) product was assessed using a Qubit 4 Fluorometer and the Qubit dsDNA HS Assay Kit (Invitrogen) and the quality was evaluated using the Agilent Tapestation 4150 and the High Sensitivity D1000 assay kit, according to manufacturers' instructions. This was followed by DNA denaturation, single-strand circularization, enzymatic digestion, enzymatic digestion product cleanup and quality control steps on the AC-PCR product, according manufacturer's instructions. The resulting ssCirDNA was used for DNB preparation and sequencing on a DNBSEQ-G400 platform at the Genomics Facility of BSRC Alexander Fleming, using a G400 App-A FCS SE100 High-throughput Sequencing Set (MGI Tech Co., Ltd.).