Effect of bantam knockdown on mRNA levels in the the γ5β′2a/β′2mp/β′2mp_bilateral Mushroom Body Output Neurons (MBONs) in Drosophila Melanogaster

In order to knockdown bantam in the γ5β′2a/β′2mp/β′2mp_bilateral Mushroom Body Output Neurons (MBONs), we expressed the bantam microRNA-sponge (or the control scrambled-sponge) under the MB011B-split-Gal4 driver. Additionally, we expressed a membrane localized GFP transgene. At ZT12-13.5, MB011B>ban-spg, GFP and MB011B>scramble-spg, GFP female fly brains were diseccted, homogenized and FACS sorted using GFP flourescence to isolate the MBONs of interest. mRNA was extracted and used to construct Illumina RNAseq libraries A modified SMART-seq2 protocol was used to generate Illumina libraries from the isolated neurons (Li et al., 2017; Picelli et al., 2014). mRNA was purified using a Dynabead mRNA kit (Invitrogen). The Poly(A)-tailed mRNAs underwent reverse transcribtion and PCR amplification (25 cycles). AMPure beads (Agencourt) were used to clean the resultant cDNA libraries, which were then processed with the Nextera XT (Illumina) protocol to generate sequencing Illumina libraries. Two biological replications were sequenced on a Next-seq 500 platform with paired-end 75bp reads and one biological replication was sequenced on a Hi-seq platform with paired-end 150bp reads. Reads were aligned to the Drosophila genome (dm6) using STAR (Dobin et al., 2013). EdgeR was used to perform DGE analysis on the aligned reads of the three biological replicates (Robinson et al., 2010).