Anti rna polymerase 2 antibody

5x10⁶ Jurkat cells were infected with NL4-3.Luc. ChIP was performed 24 h later according to Natarajan et al., 2013 [47 (link)] with the addition of a nuclei isolation step using Farnham Lysis Buffer prior to sonication with a Bioruptor Pico. Specific details are listed in S1 File. Antibodies used included anti-NELF-d (Antibody TH1L from Proteintech Group), anti-RNA Polymerase II antibody (Clone N20 from Santa Cruz Biotechnology), anti-histone H3 antibody (Product 06–599 from Millipore Sigma), and Normal Rabbit IgG (Product 12–370 from Millipore Sigma).
Primers used for the transcriptional start site include the forward primer at +30 and the reverse primer at +239. Primers used for transcriptional elongation include the forward and reverse primers within the tat gene (see S1 File).

Cells were crosslinked for 15 min using formaldehyde (1% final concentration) (Sigma, #F8775) at room temperature. Next, they were harvested and nuclei were extracted, lysed and sonicated. IP was performed using magnetic beads (Invitrogen, #100-02D) and 5 μg of antibody for each condition. Anti-RNA Polymerase II antibody was purchased from Santa Cruz (sc-899) and histone antibodies were purchased from Abcam: H3K4me3 (ab8580), H3K9Ac (ab10812), H3 (ab1791). After reverse crosslink, ChIP enriched fragments were evaluated by qPCR and reported to input DNA levels.