Yki- and Ras-induced intestinal stem cells (ISCs) overproliferation affects gene expression in the midgut of adult flies

Purpose: Activation of yki or Ras signaling pathways in ISCs causes overproliferation and ISC reprogramming. To gain insight into yki- and Ras-induced ISC reprogramming and gene expression, we performed a transcriptomic analysis of adult midgut with overexpression of yki-3SA or Ras1A in the ISCs. Overall design: Methods: To extract total RNAs for RNA-Seq experiment, we used 10 midguts dissected from both esg-Gal4, tub-Gal80ts, UAS-GFP/+ (Con) and esg-Gal4, tub-Gal80ts, UAS-GFP/UAS-Yki-3SA (Yki) and esg-Gal4, tub-Gal80ts, UAS-GFP/UAS-Ras1A (Ras) flies incubated for 8 days at 29°C. After assessing RNA quality with Agilent Bioanalyzer, mRNAs were enriched by poly-A pull-down from total RNA samples (200ng - 1ug per sample, RIN > 7 required). Then, sequencing libraries constructed with Illumina TruSeq RNA prep kit were sequenced using Illumina HiSeq2000 at the Columbia Genome Center (http://systemsbiology.columbia.edu/genome-center). We multiplexed samples in each lane, which yields targeted number of single-end 100 bp reads for each sample, as a fraction of 180 million reads for the whole lane. We use RTA (Illumina) for base calling and bcl2fastq (version 1.8.4) for converting BCL to fastq format, coupled with adaptor trimming. We map the reads to a reference Drosophila genome (flybase genome annotation version r5.51) using Tophat (version 2.1.0) with 4 mismatches (--read-mismatches = 4) and 10 maximum multiple hits (--max-multihits = 10). To tackle the mapping issue of reads that are from exon-exon junctions, Tophat infers novel exon-exon junctions ab initio, and combine them with junctions from known mRNA sequences (refgenes) as the reference annotation. We estimate the relative abundance (aka expression level) of genes and splice isoforms using cufflinks (version 2.0.2) with default settings. We test for differentially expressed genes under various conditions using DEseq. It is an R package based on a negative binomial distribution that models the number reads from RNA-seq experiments and test for differential expression.