Phosphoimager fla 7000

Total RNAs (5 μg or 10 μg) with RNA Loading Dye (NEB) were denatured for 5′ at 65°C in thermal cycler and separated by electrophoresis (Mini-Protean Tetracell BioRad apparatus) in 7% denaturing polyacrylamide gel with 8M urea in 1× Tris/borate/EDTA (TBE) solution at 100 V for 5′, then at 150 V for 50′, constantly heated in water bath at 45°C. Low Range ssRNA Ladder (NEB) was used as molecular weight standard. Gel was stained by SYBR™ Gold (Thermo Scientific) and transferred to Amersham Hybond-XL (GE Healthcare) membrane using Trans-Blot^® Turbo™ Transfer System (BioRad). Membrane was washed in 1× TBE and stored for hybridization with radioactively labelled dsDNA TR probes (Supplementary File S1B). TR probes were labelled using the DecaLabel DNA labelling kit protocol (Thermo Scientific). The membrane was hybridized overnight at 55°C with the respective [³²P-dATP]-labelled TR probe and signals were visualized using a PhosphoImager FLA-7000 (GE Healthcare). For more details about TR probes and used chemicals see Supplementary File S1C.

Telomere length analysis was performed as described in (34 ) by Southern hybridization of terminal restriction fragments (TRF) produced by digestion with Tru1I restriction endonuclease (Thermo Scientific). A membrane was hybridized overnight at 55°C with telomere probe (Supplementary File S1C). Telomere probe was synthesized by non-template PCR according to (35 (link)) and radioactively labelled with [³²P-dATP] using DecaLabel DNA labelling kit (Thermo Scientific). Telomere signals were visualized using a PhosphoImager FLA-7000 (GE Healthcare). Telomere lengths were calculated by WALTER toolset v2.0 (36 (link)) using default setup with background correction.