Miscript 2 reverse transcriptase system

Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) was performed after RNA isolation from cells using TRIzol (Invitrogen). RNA samples were DNaseI (Qiagen, 79254) treated in Figures 2, 3 and 6. cDNA was prepared in Figures 4 and 5 using Oligo(dT) and dNTPs with M-Mulv RT (NEB#M0253) and in Figures 2, 3 and 6; Supplementary Figures S4 and 5 using MiScript II Reverse Transcriptase system (Qiagen). GoTaq qPCR Master Mix (Promega A6002) was used for qPCR with primers specific to human or mouse as listed in the Supplementary Table S3. Relative quantities were calculated using the ΔΔCt method, normalized to the geometric mean of β-actin and GAPDH unless otherwise indicated.

Pulse labeling was performed using the Nascent RNA Click-iT kit (Life Technologies) according to the manufacturer's instructions. Briefly, cells were plated at 2.5 × 10⁵ per well in a 6-well plate and transfected with siRNA the following day. Seventy-two hours after transfection, ethynyl uridine (0.2 mM) was added. Media was changed after 1 h incubation and RNA was extracted 1 h later using TRIzol (Invitrogen). RNA (5 μg) was labeled with biotin (0.5 mM Biotin Azide per sample). Pulse-labeled, biotinylated RNA (500 ng) was captured using Dynabeads Streptavidin T1 magnetic beads and used as a template for cDNA synthesis using MiScript II Reverse Transcriptase system (Qiagen).