Fluorescent in situ hybridization (FISH)

FISH analysis was performed as previously described [42]. Briefly, cells were cultured on 12 mm coverslips in 24 well plates, fixed in 4% PFA, and permeabilized in 0.3% Triton for 15 min. Before hybridization, cells equilibrated in 2× SSC + 50% formamide for 10 min at 60C and probe mixture was preheated for 10 min at 95C. For hybridization, 200 mL of hybridization buffer and 27 mL of probe mixture were added to each coverslip and cells were incubated for 1 h at 60C. Following hybridization, cells were incubated with 2× SSC + 50% formamide for 20 min at 65C, then again with fresh 2× SSC/50% formamide for 15 min at 65C, and finally with 1× SSC + 40% formamide for 10 min at 60C. Cells then underwent a series of washes at RT: 3× quick washes with 1× SSC, 2 × 5 min washes with 1× SSC, a 5 min wash with Tris-buffered saline, and a 5 min wash with Tris–glycine buffer. Cells were post-fixed in 3% PFA and then incubated with blocking buffer for 1 h at RT. Cells incubated with primary antibodies diluted in immunofluorescence buffer overnight at 4C and secondary antibodies diluted in immunofluorescence buffer for 20 min at RT. After secondary antibody incubation, cells were sequentially washed with immunofluorescence buffer, Tris-buffered saline, Tris–glycine buffer, 2 mM MgCl2 in PBS, and PBS. Coverslips were mounted with ProLong Gold Antifade mounting media with DAPI (Invitrogen) and blinded. Hybridization buffer consisted of 40% formamide, BSA (2 mg/mL, Roche), 1 mM ribonucleoside vanadyl complex (Sigma-Aldrich), 10 mM NaPO4, and 1× SSC. Probe mixtures for detection of C9 RNA foci, consisted of 1 mL salmon sperm (10 mg/mL, Thermo Fisher Scientific), 0.5 mL E. coli tRNA (20 mg mL, Thermo Fisher Scientific), 25 mL 80% formamide, and 0.4 mL 25 mM 50 digoxigenin (DIG)-labeled CCCCGGCCCCGGCCCC locked nucleic acid probe (Exiqon, batch 620574). Probe mixture for detection of poly (A) RNA, consisted of 1 mL salmon sperm (10 mg/mL, Thermo Fisher Scientific), 0.5 mL E. coli tRNA (20 mg mL, Thermo Fisher Scientific), 25 mL 80% formamide, and 0.8 mL 25 mM 30 digoxigenin (DIG)-labeled polyT(25) locked nucleic acid probe (Exiqon, lot 237566815). Tris-buffered saline comprised of 50 mM Tris and 15 mM NaCl solution, pH 7.4. Tris–glycine buffer comprised of 0.75% glycine and 200 mM Tris solution, pH 7.4. Blocking buffer consisted of 1% normal donkey serum (Jackson Immunoresearch) and 5% heat-shocked BSA in Tris-buffered saline. Immunofluorescence buffer consisted of 2% heat-shocked BSA in Tris-buffered saline. DIG-labeled probe was detected with a fluorescein-conjugated sheep anti-DIG antibody (1:250, Roche). All buffers were made with RNase-free water or PBS.