Total RNA from male and female whole embryos (2 hours 2 days old), whole fourth instar larvae, whole pupae, and whole adult flies was extracted individually from each in 2-3 replicates using TRIzol (Invitrogen/Thermofisher). RNA quantity and purity were measured with the NanoDrop (ThermoScientific) and Qubit (ThermoFisher). Total RNA was treated with DNase (Qiagen) and subject to RNeasy column clean up (Qiagen). The cleaned total RNA quantity and purity were checked using the NanoDrop and Qubit. RNA integrity was evaluated on 1.1% formaldehyde 1.2% agarose gels. Poly-A RNA was enriched using Oligo-dT DynaBeads (LifeTechnologies). Qubit was used to measure the quantity of poly-A RNA. Poly-A RNA was fragmented with the Magnesium Fragmentation Module (New England Biolabs, NEB) for 3 minutes at 94C, which was selected after optimizing for conditions for 200-500 bp fragments as determined by the Bioanalyzer. Fragmentation reactions were cleaned up with RNeasy columns (Qiagen). First strand synthesis (FSS) was performed with SSIII (Invitrogen). Briefly, 1 l Random Primer (3 g/l), 1 10 mM dNTP mix, and 10 l fragmented RNA were incubated at 65C for 5-10 minutes and transferred to ice for 5 minutes. A mix of 4 l 5X First Strand Synthesis (FSS) Buffer, 1 l 0.1 M DTT, 1 l Murine RNase Inhibitor, 1 l 0.5 g/l Actinomycin D, and 1 l SSIII (200 units) was added to the mixture of RNA, dNTPs, and Random Primers. This was incubated in the thermocycler at 25C for 5 minutes to anneal the random primers, 50C for 60 minutes to extend from the random primers, and 70C for 15 minutes to inactivate SSIII. The reaction was cleaned up with AMPure beads (Beckman Coulter) using a ratio of 2.0. For Second Strand Synthesis (SSS), a mixture of 10 mM each of dATP, dCTP, dGTP, and 20 mM of dUTP (instead of dTTP) was made. For a single reaction, 64 l of cleaned FSS cDNA:RNA in Ultra Pure Water, was combined with 4 l ACGU mix, 8 l of NEB dNTP-free SSS Reaction buffer and 4 l NEB SSS Enzyme mix from the SSS module. The reaction was incubated for 1 hour at 16C, then cleaned with AMPure beads using a 1.0x ratio to begin eliminating DNA smaller than 200 bp, and quantified with the Qubit. There was typically 100-200 ng at this step. The double-stranded cDNA was then subject to NEBNext End Repair (NEB), and cleaned with AMPure beads using a 0.9x ratio to deplete DNA < 300 bp. The End-Repaired cDNA was then dA-tailed (NEBNext), and cleaned with AMPure beads using a 0.9x ratio. For adapter ligation, 38 l of DNA was combined with 10 l 5X NEBNext Quick Ligation Reaction Buffer, 1 l NEB Adaptor for Illumina sequencing, and 2 l Quick T4 Ligase. The reaction was incubated at room temperature for 15 minutes, then cleaned with a 0.9x ratio of AMPure beads. The library was then size-selected with AMPure beads before proceeding to the PCR step. To obtain adapter-ligated fragments in the 300-600 bp range, the DNA was first incubated with 0.6x AMPure beads by adding 60 l AMPure beads to 100 l DNA in UPW. The beads were pelleted on a magnet and the supernatant containing DNA smaller than approximately 600 bp was transferred to a new tube (DNA longer than 600 bp stayed on the beads). To make the final ratio 0.9x to select for DNA > 300 bp on the beads, 30 l more AMPure beads was added to the 160 l supernatant. From there the AMPure clean-up proceeded as normal. USER enzyme digestion (NEB) to cut the DNA at uracils (in the second strand and in the NEB hairpin adapters) and PCR were then performed as follows: 20 l of cleaned DNA was combined with 25 l NEBNext High-Fidelity 2X PCR Master Mix and 3 l NEBNext USER enzyme. This reaction was incubated for 15 minutes at 37C to ensure uracil cutting occurs before addition of primers. Then 1 l indexed primer (NEBNext) and 1 l universal primer were added. The reaction was put in the thermocycler for 37C for 15 minutes to ensure USER digestion went to completion, followed by 98C for 30 seconds and 12 cycles of: 98C for 10 seconds, 65C for 30 seconds, 72C for 30 seconds. The PCR products at this stage are approximately 122 bp longer than the target insert size. We adjusted AMPure ratios accordingly for a final clean up and size-selection. The PCR reactions were cleaned with 0.85x AMPure beads to deplete DNA smaller than 350 bp. The DNA was eluted in 100 l UPW and AMPure size selection was initiated by incubating with 55 l AMPure beads (0.55x) to precipitate DNA longer than 650 bp onto the beads. The beads were pelleted on a magnet and the 155 ul of DNA shorter than 650 bp in the supernatant was transferred to a new tube where another 30 l of AMPure beads was added for a final ratio of 0.85x. The AMPure procedure then continued as normal to obtain DNA > 350 bp. The estimated insert sizes at this step was 230-530 bp. DNA samples were quantified with Qubit and purity was measure by NanoDrop. There was typically 600 ng at the end of this protocol. Bioanalyzer traces suggested the mean estimated fragment sizes was around 420 bp putting the mean insert sizes near 300 bp.