RNA IP

Matched donor PB-Ts or T_EXP were UV-crosslinked with 150 mJ/cm² at 254 nm using a UV Crosslinker (Spectroline) and were lysed with lysis buffer containing 100 mm KCl; 5 mm MgCl₂; 10 mm HEPES, pH 7.0; 0.5% Nonidet P-40 detergent supplemented with fresh 1 mm DTT; 1,000 units/ml RNAsin (Promega); and Mini Protease Inhibitor Cocktail (Thermo Fisher Scientific). The post-nuclear cytosolic content was collected and removed for input samples (10%). The remaining lysates were immunoprecipitated with 10 μg anti-GAPDH Abs (Life Technologies) or with control mouse IgG Abs (The Jackson Laboratory) using a Pierce Crosslink IP Kit (Thermo Fisher Scientific). The rest of the immunoprecipitated complexes were digested with 30 μg proteinase K. The immunoprecipitates and input samples were subjected to RNA extraction using TRIzol LS Reagent (Life Technologies). RNA isolates were used for first-strand cDNA synthesis using a SuperScript VILO cDNA Synthesis Kit (Life Technologies). cDNA was used for qPCR quantification, with HIF1α or β-actin as a control. The Ct for each RNA IP sample was normalized to that of total input to account for the differences in sample preparation using the following calculation: ΔCt (normalized) = Ct (sample) – (Ct [input] – log₂ [10]).

The ΔCt (normalized) of the GAPDH IP sample was further normalized to the IgG IP sample as follows: ΔΔCt = ΔCt (normalized)^GAPDH – ΔCt (normalized)^IgG.

The immunoprecipitation fold enrichment above the sample-specific background was calculated as follows: fold enrichment = 2^(–ΔΔCT).