PRJNA563538
Wts/Lola/Yki-induced intestinal stem cell (ISC) overproliferation affects gene expression in fly midgut
Purpose: Loss of wts or lola, or overexpression of yki in either precursors or ECs causes ISC overproliferation. To get better understanding of the possible epistasis interaction of Wts/Lola/Yki in midgut homeostasis maintenance, we performed a transcriptomic analysis of adult midgut with expression of wts RNAi, lola RNAi or yki.Methods: To extract total RNAs for RNA-Seq experiment, we used 30 midguts dissected from Actin-Gal4; tub-Gal80ts (Actints, control), Actints > wts RNAi (wts RNAi), Actints > lola RNAi (lola RNAi), Actints > yki (yki) flies incubated for 5 days at 29°C per sample. Invitrogen™ TRIzol™ Reagent (15596026, Invitrogen) is used to isolate total RNA according to the User Guide.After assessing RNA quality with Agilent Bioanalyzer, mRNAs were enriched by poly-A pull-down from total RNA samples. Then, sequencing libraries constructed with Illumina TruSeq Stranded mRNA Sample Prep Kit were sequenced using Illumina HiSeq X ten. We sequenced about 26M paired-end 150 bp reads for each sample. After quality control with fastqc (version 0.11.8), we mapped the reads to a index of reference Drosophila melanogaster genome (BDGP6) using Hisat2 (version 2.1.0).After converting sam files to bam files with samtools (version 1.9), the RNA-seq alignments were assembled into transcripts using StringTie (versions 2.0). The differential expression was tested using R package ballgown (version2.16.0).