Ultrahyb oligo hyb

Total RNA was extracted from cell lysates by GTC-Phenol extraction. 10 μg total RNA was separated on an 8% polyacrylamide TBE-Urea gel and transferred to a nylon membrane (HyBond N+, GEHealthcare, RPN1210B) by electroblotting for 4 hr at 50 V. Membranes were pre-hybridised in 10 ml of UltraHyb Oligo Hyb (Thermo Scientific, AM8663) for 1 hr and probed with ³²P-labeled DNA oligo at 42°C for 12–18 hr in a hybridization oven. The sequences of the probes used for Northern blot detection are detailed in Supplementary file 10. Membranes were washed twice with 2xSSC + 0.5% SDS solution for 10 min and visualized using a Phosphor imaging screen and FujiFilm FLA-5100 Scanner (IP-S mode). For detection of highly abundant species (5S rRNA) autoradiography was used for exposure.

Total RNA was extracted from cell lysates by GTC-Phenol extraction. For large RNA fragments, 10 μg of total RNA was resolved on a 1.25% BPTE-gel (pH 7) and transferred to a nylon membrane (HyBond N+, GEHealthcare, RPN1210B) by capillarity. For short RNA fragments, 10 μg total RNA was separated on an 8% polyacrylamide TBE-Urea gel and transferred to a nitrocellulose membrane by electroblotting for 4 h at 50 V. Membranes were pre-hybridized in 10 ml of UltraHyb Oligo Hyb (Thermo Scientific, AM8663) for 1 h and probed with ³²P-labelled DNA oligo at 42°C for 12–18 hours in a hybridization oven. The sequences of the probes used for Northern blot detection are detailed in Supplementary Table 4. Membranes were washed twice with 2xSSC +0.5% SDS solution for 10 minutes and visualized using a Phosphor imaging screen and FujiFilm FLA-5100 Scanner (IP-S mode). For detection of highly abundant species (5S rRNA), autoradiography was used for exposure.