title: Illumina HiSeq 2000 sequencing; Transcriptional profiling of esg+-midgut progenitor cells expressing esgRNAi or UAS-esg AND of whole midgut tissue from flies overexpressing UAS-esg in enterocyte (EC) cells
design: Transcriptional profiling of esg+-midgut progenitor cells expressing esgRNAi or UAS-esg AND of whole midgut tissue from flies overexpressing UAS-esg in enterocyte (EC) cells
library_construction_protocol: esg-Gal4ts flies were crossed to either w1118 (esgtsCon), UASt-esg (UAS-esg) or esgRNAiJF03134 (esgRNAi) and shifted for 24 hours to 29 degrees C to induce transgene expression. 80-90 midguts were prepared for each genotype. For midgut cell dissociation for RNA-Seq, 1 mg/ml of Elastase were added per sample and guts incubated for 1 h at 27  degrees C, shaking at 600 rpm. To help dissociation, samples were pipetted vigorously every 15 min. For FACS profiling, tissue dissociation was performed by treatment with 7.5 mg/ml of Collagenase for 45 min followed by addition of 50 ul of 10X Trypsin/EDTA (Sigma) for 15-20 min. For RNA-Seq, a minimum of 20,000 GFP+ cells/condition was sorted on a FACS Aria II Flow Cytometer (BD Biosciences) using a 70 um nozzle and FACS Diva software. Intestinal cells obtained from w1118 animals where used to control for auto-fluorescence and set sorting gates. Dead cells were excluded from the sorting procedure by staining with 3 ul of Propidium Iodide (1 mg/ml, Life Technologies/Molecular Probes). RNA was isolated from sorted cells using the Arcturus PicoPure RNA Isolation Kit (Life Technologies). mRNA was amplified using the Arcturus RiboAmp HS Plus Amplification Kit (Life Technologies). Amplified RNA integrity was determined with an Agilent 2100 Bioanalyzer before library generation. cDNA libraries were generated using the NEBNext Ultra RNA Library Prep Kit for Illumina (NEB), and NEBNext Multiplex Oligos for Illumina (NEB) were used for multiplexing.
attributes.tag: Experimental Factor: genotype
attributes.value: UAS-esg overexpression
attributes.tag: LIBRARY_STRAND
attributes.value: not applicable