Control_Female_03Feb
To extract high-molecular weight DNA for sequencing, flies were homogenized in BL Buffer (Omega BioTek, Inc.) using a motorized pestle system with sterile, single-use pestles, and DNA was extracted using the E.Z.N.A. Tissue DNA Kit (Omega Bio-Tek, Inc.). High-molecular weight DNA was then sheared to approximately 500 base pairs using a Covaris ME220 Focused-ultrasonicator (Covaris,Inc.). Sheared DNA was quality checked using a High Sensitivity D1000 Screen Tape (Agilent Technologies, Inc.). DNA libraries were then prepared using the NEBNext UltraTMII DNA Library Prep Kit for Illumina with AMPure XP purification beads and NEBNext Multiplex Oligos for Illumina. Individual libraries were quality checked using a High Sensitivity D1000 ScreenTape and qPCR to determine amplifiable concentration. Libraries were then pooled and quality checked using a High Sensitivity D1000 ScreenTape and qPCR to obtain a final amplifiable concentration of approximately 5 nM for sequencing.  Sequencing was performed using 150 paired-end chemistry on an Illumina NovaSeq6000. Trim Galore! v0.6.0 and FastQC v0.4.4 were used for quality trimming and adapter removal of raw reads, retaining a minimum sequence length of 50 and quality threshold of 10. Trimmed reads were aligned to the D. melanogaster genome(release 6.29) mapping algorithm BWA-MEM in BWA (version 0.7.17-r1188). Files were filtered for read pairs at a threshold mapping quality of 20 and converted the SAM files to BAM format using SAMtools. Picard Tools (version 2.22) assigned desired read groups to the header less BAM files in the output and removed potential PCR duplicates.