Normal mouse serum

We developed a staining panel to sort PBMC into four subtypes; CD3+ CD4+ T cells, CD3+ CD8+ T cells, CD3-CD19+ B cells, and CD3-CD56+ NK cells. Table 1 provides details for each of the antibody/fluorophore conjugates used in this panel. An antibody cocktail was developed consisting of optimal lot dilutions for signal to noise of anti-CD14 FITC, anti-CD235a FITC, anti-CD8 PE, 7-AAD, anti-CD56 PE-Cy7, anti-CD4 Pacific Blue, anti-CD19 APC, and anti-CD3 AlexaFluor700 (Table 1). Antibody cocktails were prepared in a BSL2 containment hood with the light off to minimize antibody-conjugated fluorochrome degradation in stock vials over time and to maintain sterility.
Cells to be stained were first blocked in 2% normal mouse serum (#M5905, Sigma, Saint Louis, MO) in DPBS/1% BSA for 5 min at room temperature. Cells were then spun at 300 × g for 10 min, decanted and incubated with antibody cocktail (10 μl per 1 × 10⁶ cells) on ice in the dark for 80 min. Cells were washed twice with 2 ml of PBS/1% BSA, spun at 300 × g for 10 min, resuspended in 200 μl of PBS/1% BSA and filtered through a 35 μm cell strainer into a 5 ml Falcon polystyrene test tube (#352235, BD Biosciences, San Jose, CA).

Thirty to forty million cj319-WT cells were transfected with Control or HSUR 2 ASO as described above and used to prepare whole cell extracts in 0.35–0.4 ml of NET-2 buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Nonidet-40) containing cOmplete^™ protease inhibitors, 1 mM PMSF and 5 μl of RNAseOUT^™ (ThermoFisher Scientific). Extracts were sonicated on ice three times for 10 sec each. Lysates were cleared by centrifugation at 16,000 × g for 10 min at 4°C. Aliquots equal to 10% of each lysate were saved and stored immediately in 1 ml of TRIzol. Rabbit anti-mouse IgG (ThermoFisher Scientific) was immobilized on Protein A Sepharose 4 Fast Flow beads (GE Healthcare) in bulk overnight at 4°C and aliquoted into corresponding tubes before the last wash to guarantee that all samples were incubated with the same amount of antibody. The remaining 90% fraction of each lysate was incubated with 5 μl of anti-PAN-Ago antibodies -or normal mouse serum (Sigma Aldrich)- and 30 μl of Rabbit anti-mouse IgG with continuous rotation for 4 hours at 4°C. Samples were washed four times with NET-2 buffer containing protease inhibitors and the RNA immunoprecipitation “RIP” fractions were collected by centrifugation and stored immediately in TRIzol at −80°C.