2.10. Whole Transcriptome Sequencing and Analysis

The Illumina NGS sequencing was performed at the Weizmann Crown institute for Genomics, Rehovot, Israel. Briefly, libraries were prepared from RNA samples, extracted from bodies and heads of 12-day-old flies using in house protocol. For each line (w1118, GBA1a^m/+, GBA1a^m/m, GBA1b^m/+ and GBA1b^m/m), triplicates of fifty flies were used. Samples were sequenced on two lanes of Illumina HiSeq 2500 machine, using the Single-Read 60 protocol. The output was ~15 million reads per sample. Reads were trimmed using cutadapt ((https://cutadapt.readthedocs.io/en/stable/) and mapped to Drosophila melanogaster BDGP6 genome (downloaded from Ensembl genomes) using STAR v2.4.2a [23] [(https://code.google.com/archive/p/rna-star/;) default parameters]. Counting proceeded over genes annotated in Ensembl release 31 (http://metazoa.ensembl.org/Drosophila_melanogaster/Info/Index) using htseq-count (https://htseq.readthedocs.io/en/release_0.11.1/) (intersection-strict mode). Differential expression analysis was performed using DESeq2 [24] (doi:10.1186/s13059-014-0550-8) with the betaPrior, cooksCutoff and independentFiltering parameters set to False. Raw P values were adjusted for multiple testing using the procedure of Benjamini and Hochberg [25]. Pipeline was constructed using Snakemake (https://snakemake.readthedocs.io/en/v3.9.1/). Gene lists were created by filtering the genes based on an absolute linear fold change ≥2, p ≤ 0.05, and reads ≥30. To view gene lists as a heat map, the Morpheos tool was used (https://software.broadinstitute.org/morpheus/). Gene lists were analyzed for enriched pathways using the Gene Ontology tool (http://geneontology.org).