GSM5494474: pGMR_bon; yki-RNAi_rep3; Drosophila melanogaster; RNA-Seq
Dissected pupal eyes were immediately transferred to 100 μl of TRIzol (Thermo Fisher Scientific, Cat# 15596026) and kept at RT for 5 min to lyse. Samples in TRIzol were stored at -80°C until 60 pupal eyes for each genotype were collected. All TRIzol samples for each genotype were then thawed and combined, and the volume was brought up to 500 μl per sample. Total RNA was extracted twice by adding 100 μl of chloroform, vigorously shaking for 15 secs, incubating at RT for 3 min, centrifuging at 11,000 rcf at 4°C for 15 min, and collecting the aqueous phase. The total RNA was then precipitated by adding 1 volume of 70% ethanol (v/v in RNase-free water), and purified with RNeasy Mini Kit (QIAGEN, Cat# 74104, 79254). The RNA-seq libraries were prepared from 100 μg of total RNA per genotype with NEBNext Ultra II RNA Library Prep Kit mRNA Isolation Module (NEB, Cat# E7775S, E7490S, E7500S). Quality control and sequencing of the RNA-seq libraries were performed by GENEWIZ, Inc. using Agilent TapeStation and Illumina HiSeq 4000 with a 2x150 paired-end configuration.