Rabbit anti ha tag antibody

Cells were seeded (5 × 10⁶) in to 10 cm tissue culture plates. The following day cells were transfected with either 10 μg HA-Nr4a1 or 10 μg Nr4a1 and collected 48 h later. ChIP assays were performed using the Zymo-Spin ChIP Kit (Cambridge Bioscience) with the following modifications. Chromatin shearing was performed by sonication (4 cycles of 30 s on ice) using a MSE Soniprep 150 probe sonicator. Immunoprecipitations were performed with 10 μg of sheared chromatin as input and 1 μg of rabbit anti-HA tag antibody (Abcam, ab9110) at 4°C overnight with rotation. The complexes were captured with 15 μl of Dynabeads Protein G (Thermo Fisher Scientific, Waltham, MA, USA). The ChIP enrichment for the mouse Creb3l1 promoter DNA was determined by RT-PCR using primers NBRE2 (5′-CATTCCCCACAAGTTCCTGC-3′ and 5′-TGTTTGCCTGCCTCGTAAAG-3′) and NBRE3 (5′-TGACTCTCCACCTGACCTTC-3′ and 5′- TCAGTGACGCACAGGAAGAA-3′).

Bloodstream form T. brucei cultures were fixed and processed as previously described 9. Pre‐cleared chromatin (5 × 10⁷ cells per IP) was incubated with each antibody (90 μg anti‐SNF2PH, 6 μg anti‐TbRPAI, 6 μg of rabbit anti‐HA tag antibody (abcam), and 90 μg of an unspecific antiserum). The immunoprecipitated products were reverse crosslinked, and the extracted DNA was analyzed by quantitative PCR (qPCR). For ChIP‐seq analysis, the protocol was scaled for a final concentration of ~5 ng of immunoprecipitated DNA. To compare the amount of DNA immunoprecipitated to the total input DNA, 10% of the pre‐cleared chromatin saved as input was processed with the eluted immunoprecipitated products before the crosslink reversal step. Quantitative PCR was performed using SYBR green Supermix (Bio‐Rad) in a CFX96 cycler (Bio‐Rad). IP percentages were determined as previously described 9. At least three independent experimental assays were displayed and analyzed by Student's t‐test. A detailed primer list is detailed in Appendix Table S4.