Click it nascent rna capture kit

Analysis of mRNA half-lives was performed by EU pulse-labeling of RNA using the Click-iT Nascent RNA Capture Kit (ThermoFisher). OP9 cells were seeded in 10 cm tissue culture dishes, grown to confluence, and then induced to differentiate with a modified DMI cocktail protocol. Briefly, cells were stimulated with the typical DMI cocktail for 24 hours. Then half the volume of differentiation media in each plate was removed and combined. To avoid stimulation by fresh media, 5EU was directly added to the combined differentiation media to a final concentration of 200 µM. The remaining half of differentiation media in each plate was then aspirated and replaced with media containing 5EU and incubated for another 24 hours. At 48 hours, all plates were washed 3 times with fresh culture media and replaced with 5EU-free culture media containing only insulin. Cells were harvested at the indicated time points and total RNA was isolated using TRIzol reagent (ThermoFisher). EU-labeled RNAs were biotinylated and captured on streptavidin coated magnetic beads using the Click-iT Nascent RNA Capture Kit (ThermoFisher), following the manufacturers protocol. RT-qPCR was performed on the captured RNA to assess mRNA half-lives.

Murine B220⁺ splenocytes or human cells transfected using Lipofectamine 2000 (Invitrogen) with 50 nM siRNAs targeting HSF1 (Hs_HSF1_6: 5’-GCUUCGUGCGGCAGCUCAATT-3’, Hs_HSF1_9: 5’-GGUUGUUCAUAGUCAGAAUTT-3’, Qiagen) or a non-targeting control (Stealth RNAi medium GC duplex, Invitrogen) for 48 h were heat shocked at 43 °C for 2 h followed by recovery at 37 °C for 2 h while simultaneously pulsed with 0.2 mM ethynyl uridine (EU). EU-labeled RNA was captured with the Click-It Nascent RNA capture kit (Molecular Probes). Briefly, RNA was biotinylated, conjugated to streptavidin beads, and converted to cDNA using the SuperScript III first-strand synthesis system (Invitrogen). A fraction of the undiluted cDNA was used in the QPCR and detected by fast SYBR Green (Quanta Biosciences) on the 7900HT Fast Real-Time PCR System (Applied Biosystems).

RNA was isolated using TRI reagent (Sigma) and samples were reverse transcribed using qScript cDNA SuperMix (Quantas). Quantitative real-time PCR was performed on an Eppendorf Realplex^{2 (link)} mastercycler using SYBR green (Quantas). Specific primers used are listed in Supplementary Table 2. For determination of DICER mRNA half life cells were treated with 5 μg/ml actinomycin D (Sigma) for the indicated times. De novo RNA synthesis was measured using the Click-iT Nascent RNA Capture Kit (Molecular Probes). Briefly, MCF7 and HMLER cells were pulse labeled with 0.2mM 5-ethynyl Uridine for 1 hour during aerobic conditions or 24 hrs hypoxia (0.2% O₂) after which RNA was isolated as described above. Nascent RNA was captured using magnetic streptavidin beads, reverse transcribed and analyzed by quantitative real-time PCR. For determination of pri-miRNA and mature miRNA levels the following assays from Applied Biosystems were used: pri-miR200a (Hs03303376_pri), pri-miR200b (Hs03303027_pri), pri-miR429 (Hs03303727_pri), pri-miR200c (Hs03303157_pri), pri-miR141 (Hs03303157_pri), pri-miR210 (Hs03302948_pri), hsa-miR200a (000502), hsa-miR200b (002251), hsa-miR429 (001024), hsa-miR200c (002300), miR141 (002145), hsa-miR103 (000439), hsa-miR107 (000443), hsa-miR210 (000512), RNU44 (001094), RNU48 (001006).

For Nascent RNA assays, 400 WIDs nub-GAL4/+; UAS-GFP/+ were dissected and disaggregated as described above. Click-IT® Nascent RNA Capture Kit from Molecular Probes (C10635) was used according to the manufacturer’s instructions. Briefly, disaggregated cells were incubated with 0.5 mM 5-ethynil uridine (EU) in Schneider’s Insect Medium for 1 h at room temperature. Total RNA was extracted and biotinylated with 0.25 mM biotin-azide for 30 minutes at room temperature. Biotinylated RNA was precipitated overnight at −80°C and purified with Streptavidin conjugated beads for 30 minutes at room temperature. Nascent RNA was eluted in 0.1 % SDS 5 minutes at 99°C and retrotranscription was carried out as described above. Four biological replicates were performed. Primers used for Real-Time PCR are listed in Supplementary Table 11.