TT-seq

TT-Seq in S2 cells was performed largely based on protocol described in Schwalb et al. (2016), with some adaptations for use in Drosophila cell culture. Briefly, 35 million cells were plated in 10 cm dishes and 700 ug of dsRNA was added per dish. 2–3 replicates per condition were run in parallel: dsWhite, dsMtor and dsMOF. KD proceeded for 72 hours and was then repeated with 700 ug dsRNA per condition. After 48 hours, cells were harvested as follows: 50 mM 4-thiouridine (4sU) (Sigma, T4509) stock solution in H2O was prepared ahead of time and stored at 4C protected from light. Dishes were treated and harvested in manageable batches of 3 by replicate to keep timing windows precise. For each plate, 500 uM of 4sU was added in dim light. Cells were incubated for 5 mins then collected. Cells were directly centrifuged after 20 mins at 1,500 RPM for 3 min at RT. Supernatant was removed and 1mL of Trizol was added per 20 million cells, samples were resuspended, rotated at 4C for 15 minutes in the dark for homogenization, flash frozen on dry ice and stored at −80C. Mammalian cells used for spike in (IMR90 cells here) were treated identically to S2 cells to obtain enough RNA for a 5% spike in. Total RNA was isolated using standard phenol/chloroform extraction, RNA was fragmented on a Covaris sonicator (300 ug S2 RNA plus 15 ug mammalian spike in per sample), using the following settings: 1 burst: 30 s ON / 30 s OFF at high settings (200 cycles/burst, Peak Incident Power 140 W, Duty Factor 5%). Fragmentation of RNA was assessed on formamide-agarose gels, revealing a smear roughly between 1.5 kb – 200bp, showing significant depletion of rRNA band(s). RNA was next denatured at 65C for 10 minutes. Samples were split in two into batches of 150ug RNA each and biotinylated with 200 ug of EZ-link HPDP Biotin (Thermo Fisher Scientific 21341), stock at 2mg/mL in DMF) in 1X Binding Buffer (100mM Tris pH7.5, 10mM EDTA pH 8.0) and 30% vol/vol DMF with a total reaction volume of 1mL, rotating in the dark at RT for 2 hr. Biotinylated RNA was then immediately precipitated with the addition of chloroform, followed by centrifugation to isolate the aqueous layer. 1/10 volume of 5 M NaCl and 1 volume of isopropanol were then added. This was spun at 15,000 rpm at 4C for 30 mins to pellet biotinylated RNA. Pellet was washed in cold 75% ethanol, spun again for 5 mins, supernatant removed without allowing pellet to dry, and RNA was resuspended in 100 uL H2O. To separate biotinylated RNA from unlabeled as follows: 100ul of Invitrogen Magnetic Streptavidin Beads (Invitrogen 65002) were washed 2x with 2 vol Wash Buffer (WB: 100mM Tris pH 7.5, 10mM EDTA pH 8, 1M NaCl, 0.1% Tween-20),and resuspended in 1 vol WB. 200uL biotinylated RNA was incubated at 65C for 10 minutes, placed on ice for 5 minutes, mixed with 100ul of prepared streptavidin beads, and incubated at 4C for 15min on rotor. Samples were placed on magnetic rack for 3 min, washed 3x with 900uL 65C WB, 2x with 900ul RT WB, re-suspended in 50ul 1X Turbo DNase Buffer, 1ul of DNase was added and samples were incubated at 37C for 30 minutes. Samples were washed 2x with 900uL RT WB, eluted twice in 100ul RT 100mM DTT, incubated 5 minutes, transferred to magnetic rack and pooled. RNA was then purified using SPRI select beads (in a non-size selecting manner using 50uL beads, 100uL sample, 130ul bead buffer (20% PEG-8000, 2.5 M NaCl, 10 mM Tris HCl pH7.5, 1 mM EDTA, 0.05% Tween-20) and 270ul isopropanol). Beads were washed 2x with 500ul RT 80% EtOH, eluted in 10ul TE pH7.4 and stored at −80C. Quality of labeled RNA was then assessed on a Bioanalyzer RNA Chip. Final concentration of labeled RNA purified was ~200 ng per sample. Lastly, samples were then processed with the same library protocol described for RNA-Seq in salivary glands, minus Poly A+ selection.