GSM6259854: Intestine, esgTS, Rep 3; Drosophila melanogaster; RNA-Seq
Gastrointestinal (GI) tracts of 100 adult female flies (per replicate) were dissected in ice-cold 1xPBS. Then cells were dissociated by treating intestines with 1mg/ml elastase at 27oC for 1 hour with agitation. Subsequently, ~30,000-50,000 GFP+ intestinal progenitor cells were sorted using a BD FACSAriaTM II flow cytometer equipped with a 100µm nozzle at the IUB Flow Cytometry Core Facility. For CLIP-seq analysis, dissected intestines were homogenized in lysis buffer and subjected to immunoprecipitation using SWM-coated magnetic beads. A portion of the total extract was used as control for this analysis. Total RNA was prepared using the TRIzol® LS reagent (Ambion). The rRNA-depleted libraries were prepared using the Ovation® SoLo RNA-seq system (Part No. 0502 including Parts 0407 and S02240) following manufacturer's instructions. The quality and quantity of final libraries were assessed using Agilent 2200 TapeStation and KAPA Library Quantification Kit, respectively. The rRNA-depleted RNA-seq/CLIP-seq libraries were prepared using the Ovation® SoLo RNA-seq system (Part No. 0502 including Parts 0407 and S02240) following manufacturer's instructions.