Table_2_Meiocyte Isolation by INTACT and Meiotic Transcriptome Analysis in Arabidopsis.DOCX

Isolation of nuclei tagged in specific cell types (INTACT) is a method developed to isolate cell-type-specific nuclei that are tagged through in vivo biotin labeling of a nuclear targeting fusion (NTF) protein. In our work, INTACT was used to capture nuclei of meiocytes and to generate a meiotic transcriptome in Arabidopsis. Using the promoter of AtDMC1 recombinase to label meiotic nuclei, we generated transgenic plants carrying AtDMC1:NTF along with biotin ligase enzyme (BirA) under the constitutive ACTIN2 (ACT2) promoter. AtDMC1-driven expression of biotin-labeled NTF allowed us to collect nuclei of meiocytes by streptavidin-coated magnetic beads. The nuclear meiotic transcriptome was obtained by RNA-seq using low-quantity input RNA. Transcripts grouped into different categories according to their expression levels were investigated by gene ontology enrichment analysis (GOEA). The most enriched GO term “DNA demethylation” in mid/high-expression classes suggests that this biological process is particularly relevant to meiosis onset. The majority of genes with established roles in meiosis were distributed in the classes of mid/high and high expression. Meiotic transcriptome was compared with public available transcriptomes from other tissues in Arabidopsis. Bioinformatics analysis by expression network identified a core of more than 1,500 genes related to meiosis landmarks.特异性细胞类型细胞核标记分离技术（Isolation of Nuclei Tagged in Specific Cell Types，简称INTACT）是一种通过对核靶向融合（nuclear targeting fusion，NTF）蛋白进行体内生物素标记，以分离获取细胞类型特异性细胞核的技术。本研究中，我们借助INTACT技术分离拟南芥减数分裂细胞的细胞核，并构建了拟南芥减数分裂转录组。我们利用AtDMC1重组酶的启动子标记减数分裂细胞核，构建了同时携带AtDMC1:NTF融合基因，以及在组成型ACTIN2（ACT2）启动子驱动下表达生物素连接酶（biotin ligase enzyme，BirA）的转基因植株。在AtDMC1的驱动下，生物素标记的NTF蛋白得以表达，借此我们可通过链霉亲和素包被磁珠收集减数分裂细胞的细胞核。我们以低起始量RNA为模板，通过RNA-seq获取了细胞核水平的减数分裂转录组。我们根据表达水平将转录本划分为不同类别，并通过基因本体富集分析（gene ontology enrichment analysis，GOEA）对各类别转录本展开了研究。在中高表达类别中，富集程度最高的基因本体术语为"DNA去甲基化"，这表明该生物学过程与减数分裂起始具有密切关联。绝大多数已被证实参与减数分裂过程的基因，均分布在中高表达和高表达类别中。我们将所构建的减数分裂转录组与公共数据库中拟南芥其他组织的转录组进行了比对分析。通过表达网络的生物信息学分析，我们鉴定得到了1500余个与减数分裂标志性事件相关的核心基因。