Effects of formalin-fixation on FAIRE-Seq and MNase-Seq signatures in yeast

Formalin induces inter- and intra-molecular crosslinks within exposed cells. This cross-linking can be exploited to characterise chromatin state as in the FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements) and MNase (micrococcal nuclease) assays. Our team aims to optimise these assays for application in museum preserved formalin-exposed specimens. To do so, we must first understand the effect of prolonged formalin fixation on the read alignment signatures resulting from FAIRE and MNase treatment. Here we cultured yeast (Saccharomyces cerevisiae) under normal and heat-shock conditions then fixed the cells with formalin. At pre-determined fixation timepoints (15min, 1 hour, 6 hours and 24 hours), we removed aliquots of fixed cells and processed them through FAIRE and MNase protocols optimised for heavily fixed yeast cells. Alongside these samples, we collected genomic DNA and RNA from both regular and heat-shocked cultures prior to fixation. The DNA samples were prepared for sequencing using an IDT xGEN Prism library kit and sequenced on a NovaSeq S4 100bp PE flowcell.

甲醛（Formalin）可使受其作用的细胞内形成分子间与分子内交联。该交联特性可用于表征染色质状态，正如FAIRE（甲醛辅助分离调控元件，Formaldehyde-Assisted Isolation of Regulatory Elements）与MNase（微球菌核酸酶，micrococcal nuclease）实验所示。本研究团队旨在优化上述实验方法，以应用于博物馆馆藏的经甲醛处理的标本。为此，我们首先需要明确长期甲醛固定对FAIRE与MNase处理后产生的测序读段比对特征的影响。本研究将酵母（酿酒酵母，Saccharomyces cerevisiae）分别在正常培养与热激条件下培养，随后用甲醛对细胞进行固定。在预设的固定时间点（15分钟、1小时、6小时与24小时），我们收取固定后的细胞等分试样，并按照针对重度固定酵母细胞优化的FAIRE与MNase实验流程进行处理。与此同时，我们在固定前从正常培养与热激培养的菌液中分别收集基因组DNA与RNA。DNA样本采用IDT xGEN Prism文库制备试剂盒构建测序文库，并在NovaSeq S4 100bp双端测序流动槽上完成测序。