Rnaeasy animal total rna isolation kit

Total cellular RNA was prepared with RNAeasy Animal Total RNA Isolation Kit (Beyotime R0032). tRNA demethylation and reverse transcription were performed with rtStar tRNA-optimized First-Strand Synthesis Kit (Arraystar AS-FS-004). qPCR for tRNAs was performed using the nrStar Human tRNA PCR Array (Arraystar AS-NR-001-1) and Arraystar SYBR Green qPCR Master Mix(ROX+) (Arraystar AS-MR-006-5). The data were analyzed by the ΔΔCt method.

RIP was performed following a protocol slightly adapted from Vogt and Taylor 2013 [78 ]. Briefly, cells were fixed, lysed, sonicated and centrifuged to collect the supernatant, which was then incubated with anti-NXF1 or anti-XPO1 antibody overnight at 4°C with rotation. A sample incubated with IgG was included as control. Dynabeads protein G (Thermo Fisher Scientific 10003D) were added to the antibody conjugated complex and incubated for 2 hours at 4°C with rotation. Afterward, the beads were washed and incubated for 1 hour at 70°C to reverse cross-link. Immunoprecipitated RNA was extracted with RNAeasy Animal Total RNA Isolation Kit (Beyotime R0032), and analyzed by RT-qPCR with the primers listed in Table B in S1 Text.

Yeast RNA was extracted using the hot acidic phenol method, while mammalian RNA was prepared using RNAeasy Animal Total RNA Isolation Kit (Beyotime R0032). For nuclear and cytoplasmic fraction separation, 5×10⁶ mammalian cells were washed twice with ice-cold PBS, collected, resuspended in 500 μL hypotonic buffer (20 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl₂) by pipetting several times, and incubated on ice for 15 minutes. 25 μL 10% NP-40 was then added, and the homogenate was vortexed for 10 seconds at highest setting before centrifugation at 3,000 rpm at 4°C. The supernatant contained the cytoplasmic fraction, while the pellet was the nuclear fraction.
Reverse transcription was performed using BeyoRT II cDNA First Strand Synthesis Kit (RNase H-) (Beyotime D7168) with gene-specific primers, followed by qPCR using BeyoFast SYBR Green qPCR Mix (2×) (Beyotime D7260) with the primers listed in Table B in S1 Text.