Titration-based normalization of antibody amount improves consistency of ChIP-seq experiment. Titration-based normalization of antibody amount improves consistency of ChIP-seq experiment

Chromatin immunoprecipitation (ChIP) is an antibody-based approach that is most frequently utilized in research areas of chromatin biology and epigenetics. The ENCODE Consortium provided guidelines about antibody but the fundamental aspect regarding antibody titer was not addressed. Here, we introduced a simple and quick DNA-based quantification of chromatin input, allowing to normalize the antibody amount in individual ChIP reaction to the optimal titer. We further demonstrated the titration-based normalization of antibody amount generates improved assay outcome with high consistency among samples in an experiment or recurrent experiments for a broad range of chromatin input amount. Chromatin immunoprecipitation-sequencing (ChIP-seq) was performed for histone H3K27ac mark in the conditions of various titer of antibody in ChIP reactions.. Overall design: Chromatin were prepared from fixed K562 cells. Chromatin amount was aliquoted into 312, 625, 1,250, 2,500, 5,000, 10,000, and 20,000 ng in final 1 ml of ChIP buffer. 0.25 µg of anti-H3K27ac antibody was added to create the ChIP reactions with various antibody titers (the ratios between antibody amount per chromatin amount). Or normalized amounts of the antibody to the optimal titer was added into each reaction.

染色质免疫共沉淀（Chromatin immunoprecipitation, ChIP）是一种基于抗体的实验技术，最常应用于染色质生物学与表观遗传学研究领域。ENCODE联盟（ENCODE Consortium）曾针对抗体实验制定相关指导规范，但未涉及抗体滴度这一核心要素。本研究提出了一种简便快速的染色质投入量DNA定量方法，可将单次ChIP反应中的抗体用量标准化至最优滴度。我们进一步证实，基于滴度的抗体用量标准化可优化实验结果，使同一实验内的多样本或重复实验间的结果具备高度一致性，且适用于宽泛范围的染色质投入量。本研究针对组蛋白H3K27ac修饰标记，在不同抗体滴度的ChIP反应条件下开展了染色质免疫共沉淀测序（Chromatin immunoprecipitation-sequencing, ChIP-seq）实验。实验设计概述：从固定后的K562细胞中制备染色质，将染色质总量分装为312、625、1250、2500、5000、10000及20000 ng，最终体系为1 ml ChIP缓冲液。向各组中加入0.25 μg抗H3K27ac抗体，以构建不同抗体滴度（即抗体用量与染色质质量的比值）的ChIP反应体系；或向每管反应中加入标准化至最优滴度的抗体用量。