title: RNA-Seq of Drosophila melanogaster: adult intestinal projentor cells
design: Intestinal stem cell isolation by fluorescence-activated cell sorting (FACS) was adapted from the published protocol article by Dutta et al., 2013. In brief, three biological replicates consisting of 200 fly guts per replicate were dissected in PBS-1X and then immediately transferred into a 1.5-ml microcentrifuge tube containing ice-cold diethylpyrocarbonate (DEPC)-treated water-PBS (400 l). Dissected midguts were then dissociated with 100 l elastase solution (1 mg/ml final concentration) and incubated for 1 hour at 27C in a heating block with shaking at 34  g. Dissociated cells were centrifuged for 20 min at 300  g, at 4C, and the pellets were resuspended in 500 l fresh PBS-DEPC for sorting. 1 l of 1 mg/ml propidium iodide was added, and then dissociated cells were filtered using 25-m filters to remove clumps. Cell suspension was then FACS sorted. GFP+ cells from the esgGFP>w1118  and w1118 (negative control no GFP) midguts flies were sorted out first to set the fluorescence gate. For each of the three biological replicates, about 40,000 GFP+ cells were sorted. RNA was isolated from the FACS-sorted GFP+ cells using a Direct-zol RNA Microprep kit, followed by library preparation using the QIAseq UPX3 transcriptome for next-generation sequencing paired to the QIAseq UPX 3 trans. 12 (Index 48). Libraries were pooled and quantified using QIAseq library Quant Assay kit and analyzed on a bioanalyzer (Agilent).