E coli trna

Neurons were plated on PDL-coated 28-mm glass bottom dishes (Invitrosci) and fixed 7 days later with 4% paraformaldehyde for 20 minutes at room temperature. Cells were permeabilized with cold 70% ethanol overnight at 4°C and washed the following morning for 5 minutes with wash buffer (25% formamide, 2X SSC). Neurons were stained with 40 20-nucleotide-long fluorescently labeled (Alexa-647) oligonucleotides designed against the Aag transcript overnight at 37°C in a heavily humidified chamber in hybridization buffer (100 mg/mL dextran sulfate, Sigma, D8906: 0.5 mg/mL E.coli tRNA, Sigma, R4251: 0.5 mg/mL ssDNA, Sigma D9156: 1 mg/mL Ultapure BSA, Ambion, AM2616: 10 mM VRC, New England Biolabs, S1402S: 25% formamide, Ambion, AM9342: 2X SSC, Ambion, AM9763). Finally, cells were stained with DAPI (2 μg/mL) in wash buffer for 30 minutes at 37°C before imaging. All the previous steps were done in nuclease-free solutions to avoid RNA degradation. We occasionally see faint transcripts in Aag^-/- cells due to initiation of endogenous transcription before hitting the inserted cassette that disrupts the gene.
Images were taken with a Hamamatsu ORCA-R² CCD Camera on a Zeiss Axio Observer.Z1 Microscope. 21 Z-stack images were taken, each 0.3 μm away from the previous. Images were compiled and foci per cell were counted with a MATLAB algorithm adapted from previously published methods [23 (link)].

RNA FISH was conducted as described previously [52 (link)]. Briefly, testes from 2–3 day-old flies were dissected in 1x PBS and fixed in 4% formaldehyde in 1x PBS for 30 minutes. Then testes were washed briefly in PBS and permeabilized in 70% ethanol overnight at 4°C. Testes were briefly rinsed with the wash buffer (2x saline-sodium citrate (SSC), 10% formamide) and then hybridized overnight at 37°C in the hybridization buffer (2x SSC, 10% dextran sulfate (sigma, D8906), 1 mg/mL E. coli tRNA (sigma, R8759), 2 mM Vanadyl Ribonucleoside complex (NEB S142), 0.5% BSA (Ambion, AM2618), 10% formamide). Following hybridization, samples were washed three times in the wash buffer for 20 minutes each at 37°C and mounted in VECTASHIELD with DAPI (Vector Labs). All solutions used for RNA FISH were RNase-free. Images were taken on a Leica TCS SP8 confocal microscope with a 63x oil immersion objective [numerical aperture (NA) = 1.4] and processed using Adobe Photoshop and ImageJ software. Probe sequences are listed in the S1 Table. R2 Stellaris FISH probe set was designed and synthesized by LGC Biosearch Technologies and used previously [17 (link)].

Ten specific DNA probes covering the entire 420-nucleotide (nt) sequence of full-length scaRNA2 were utilized. Each probe was 70 nt long and included a sequence antisense to scaRNA2 (30 nt) flanked with 5′- and 3′-linker sequences (á 20 nt). DNA oligomers specific for each linker sequence and containing two fluorescent labels (see Supplementary Table 3) were used for visualization.
Cells were first grown on coverslips and then fixed with 4% paraformaldehyde (PFA) (Santa Cruz Biotechnology) for 10 min. After washing in PBS and 70% ethanol, the coverslips were placed in RNA wash buffer (25% formamide (Ambion), 2x SSC (Ambion) in nuclease-free water) for 5 min and subsequently incubated overnight at 30 °C with 10 μM scaRNA2 probes (dissolved in hybridization buffer containing 10% dextran sulfate (Sigma-Aldrich), 25% formamide, 2x SSC, 0.02% BSA, 1 mg/ml E.coli tRNA (Sigma-Aldrich), and 10 mM ribonucleoside vanadyl complex (New England Biolabs)). The following day these coverslips were incubated in RNA wash buffer for 1 h at 30 °C and thereafter with 2 μM fluorescent probes in hybridization buffer for 2–3 h at 30 °C. Finally, the coverslips were exposed to RNA wash buffer for 1 h at 30 °C and washed twice with SSC before mounting with VectaShield mounting medium containing DAPI (Vector Laboratories).

Testes from 2–5 day old flies were hand-dissected in PBS and fixed in 4% formaldehyde (Methanol-free, Pierce) in PBS for 30 minutes at room temperature. Testes were washed briefly in PBS and permeabilized in 1 mL 70% ethanol overnight on a rocker in a 4°C cold room. After rinsed with 300 ul wash buffer (2X saline-sodium citrate (SSC. 300 mM NaCl, 30 mM trisodium citrate, pH 7.0), 10% formamide), testes were hybridized with 100 uL of 50 nM (for satellite repeat. Integrated DNA Technologies) or 100 nM (for all the others. Biosearch Technologies, Inc) probes in hybridization buffer (wash buffer, 10% dextran sulfate (Sigma, D8906), 1 mg/mL E. coli tRNA (Sigma, R8759), 2 mM Vanadyl Ribonucleoside complex (NEB, S1402), 0.5% BSA (Ambion, AM2618)) overnight at 37°C. After washed with 200 ul wash buffer three times for 20 minutes each at 37°C, testes were mounted in 30–40 ul of VECTASHIELD with DAPI (Vector Labs). During these procedures, sample tubes and slides containing testes with FISH probes were covered with aluminium foil to minimize photo-bleaching. Testes Images were collected with the Zeiss LSM700 confocal microscope at the Johns Hopkins University School of Medicine Microscope Facility. The FISH probes used are listed in S2 Table.