Mapping the differential distribution of the cohesin subunit Scc1 in Trypanosoma brucei brucei.. Mapping the differential distribution of the cohesin subunit Scc1 in Trypanosoma brucei brucei.

Chromatin immunoprecipitation of genomic loci in Trypanosoma brucei where Scc1 is deposited. This was achieved by deletion of one Scc1 allele and N-terminal tagging of the second Scc1 allele with a Ty1-tag. During the ChIP experiment, the DNA was crosslinked and cells were permeabilized with digitonin. DNA was fragmented by sonication with a Covaris instrument. DNA bound to Ty1-Scc1 was pulled down by using a BB2 anti-Ty1 antibody. Cross links are reversed and the DNA was purified and prepared for Illumina sequencing. Overall design: Experiment was done in triplicates, each with an input control. As a control, an input sample was included for each ChIP experiment.

本实验针对布氏锥虫（Trypanosoma brucei）中Scc1沉积的基因组位点开展染色质免疫沉淀（Chromatin immunoprecipitation, ChIP）实验。实验通过敲除一个Scc1等位基因，并利用Ty1标签（Ty1-tag）对剩余的Scc1等位基因进行N端标记完成体系构建。在实验流程中，首先对DNA进行交联处理，随后使用洋地黄皂苷（digitonin）透化细胞；采用Covaris仪器通过超声破碎法对DNA进行片段化。通过BB2抗Ty1抗体（BB2 anti-Ty1 antibody）富集与Ty1-Scc1结合的DNA。待交联反应逆转后，纯化DNA并制备用于Illumina测序（Illumina sequencing）的测序文库。实验设计概述：本实验设置三次生物学重复，每个重复均配套设置输入对照；每个染色质免疫沉淀实验均包含一份输入样本作为对照。