Anti pol 2 c terminal repeats

The wild type human Burkitt’s lymphoma cell line Ramos has been described^{30 (link), 35 (link)}. The modified reporter Ramos cell line was established by replacing the endogenous 4–34 Igh-V region with mCherry-4–34 fusion fragment (Fig1a) using recombinase-mediated cassette change (RMCE). Briefly, a LoxP flanked hygromycin resistance gene was integrated into endogenous Igh-V locus by homologous recombination. The mcherry reporter cassette was then knocked into the Igh-V locus through Cre mediated recombination³¹ . The Ramos clone used to construct this reporter line was preselected to have an undetectable level of AID protein and confirmed not to undergo SHM. Cells containing mCherry-Igh-V fusion were then transfected with AID-ER fusion protein ^{36 (link)} and clones were selected based on their capacity for undergoing SHM in a 4-OHT (Sigma-Aldrich) inducible manner. Cells were considered to have undergone SHM when they reduced their mCherry fluorescence based on flow cytometry analysis. Antibodies used in this study were anti-Pol II C-terminal repeats (Abcam), antiPol II C-terminal repeats (Serine 5 phosphorylated) (Abcam), anti-Spt5 (Santa Cruz) (1:250 dilution), anti-tubulin (Sigma-Aldrich) (1:2500 dilution).