Stratagene brilliant 2 sybr green qrt pcr master mix

RNA was extracted in TRIzol using Quick-RNA Miniprep Kit following the manufacturer’s protocol with on-column Dnase digestion (Zymo Research). RT-qPCR was performed using Stratagene Brilliant II SYBR Green QRT-PCR Master Mix (Agilent). To quantify the allelic bias, cDNA was generated by reverse transcription of extracted RNA using SuperScriptIII and the amplified for 5 cycles using targeted qPCR primers. Then the PCR product was purified by Zymo DNA Clean & Concentrator-5 and further amplified using biotinylated primers and sequenced on using Q24 Pyromark (Qiagen). All primers for qRT-PCR and pyrosequencing are listed in Table S6.

10uL Protein G Dynabeads were incubated in 1mL blocking buffer (PBS with 0.1%BSA) for 20 min at room temperature. 2ug anti-CD40L antibody (Cat#157009, Biolegend) or anti-VSV-G antibody (clone 8G5F11, Millipore sigma), or IgG antibody were added into beads with 100uL blocking buffer and rotated for 30 min at 4°C. The antibody conjugated beads were washed three times and the supernatant was removed. 30uL CD40L displayed viruses were added into beads with 30uL blocking buffer and rotated for 1 hour at room temperature. 5uL CD40L displayed virus from the same batch was prepared as input samples. The beads were washed three times and the supernatant was removed. 100uL Trizol was added into beads or input sample and subjected to RNA extraction by Zymo Quick-RNA Miniprep Kit. RT-qPCR was performed using Stratagene Brilliant II SYBR Green QRT-PCR Master Mix (Agilent).