Allele specific expression for Lectin-24A in hybrid Drosophila Genetic Reference Panel lines

In order to understand the impact of upstream indels on expression, we selected multiple DGRP lines with varying combinations of the three upstream indels and tested if parasitoid infection upregulated Lectin24A in those lines. 10 male flies from 20 DGRP lines with varying combinations of the upstream Lectin24A promoter indels were crossed with 10 virgin females DGRP437. Females were allowed to lay eggs overnight in cornmeal vials with yeast. Adult flies were removed the next day and tipped into a new vial, which served as the second biological replicate. F1 larvae in each vial developed for an additional 48 hours before they were infected with three female L. boulardi wasp strain G486 for three hours. 24 hours post infection, 10 larvae for each sample were homogenized in RTL buffer and RNA was extracted using an RNA easy miniprep kit, including optional on-column DNase treatment. cDNA was prepared using 1ul of template RNA with the GoScript Reverse Transcriptase system primed with random hexamers, according to the manufacturers instructions. For each F1 cross, genomic DNA was also extracted from eight adult flies using the Blood and Tissue DNA extraction kit. Sequencing libraries were prepared from each sample. PCR was performed on the gDNA for one of the biological replicates and cDNA from both biological replicates targeting the Lectin24A gene region on chromosome 2L between 3,717,069 and 3,717,568. that include adapter overhang sequences. Two technical replicates of each PCR reaction were performed. Libraries were pooled and sequenced on MiSeq nano 300 cycle kit, paired-end sequencing. Following sequencing, Illumina BaseSpace Sequence Hub was used to convert Binary Base Call files into FASTQ files for all reads passing filtering.