Additional file 2: of Regulatory complexity revealed by integrated cytological and RNA-seq analyses of meiotic substages in mouse spermatocytes

This file contains a mini-website of Figures S1-S6 and a link to interactive expression plots. The same mini-website is available at http://carterdev.jax.org/dtx/a1/index.html. Figure S1. Expression of substage-concordant genes. (A-H) Heat maps show gene expression of substage-concordant genes at each time point. Gene expression is shown as average log2(TPM + 1) of time point replicates. Figure S2. Expression of substage-negatively concordant genes. (A-H) Heat maps show gene expression of substage-negatively concordant genes at each time point. Gene expression is shown as average log2(TPM + 1) of time point replicates. Figure S3. Validation of RNA-seq expression pattern by qRT-PCR. Box plots (top) show gene expression detected by RNA-seq at 10 dpp and 16 dpp. Bar graphs (bottom) show gene expression detected by qRT-PCR using isolated germ cells from pooled testes from 3 mice at 10 dpp and 16 dpp. Gene expression for RNA-seq is shown as log2(TPM + 1). Gene expression for qRT-PCR is shown as fold increase relative to Actb expression. Figure S4. TF arc diagram illustrating the most significant TFs across substages for the concordant gene lists. Node color represents the substage. Node size is related to significance (larger = more significant but all are highly significant with a iRegulon NES > = 4). Width of arc corresponds to how many TFs are shared between nodes (wider = more shared TFs). A node is connected to another node if a node’s TFs are a subset of the other node’s TFs. Color of the arc (degree) is related to how many connections the node has. Degree equals the number of connections. The name of each node is an abbreviation, where an asterisk indicates multiple members of the TF family are included in the node (e.g., Rfx* for Rfx1, Rfx2, Rfx3, Rfx4) and when a node name is followed by + integer, as in “Mef2* + 1”, it indicates that there are TFs in the Mef2 family plus one other TF. All TFs associated with each node are listed in Additional file 1: Table S6. The number in parenthesis next to the node name indicates the number of substage-concordant genes associated with the TF cluster. Figure S5. TF arc diagram illustrating the most significant TFs across substages for the negatively concordant gene lists. Node color represents the substage. Node size is related to significance (larger = more significant but all are highly significant with a iRegulon NES > = 4). Width of arc corresponds to how many TFs are shared between nodes (wider = more shared TFs). A node is connected to another node if a node’s TFs are a subset of the other node’s TFs. Color of the arc (degree) is related to how many connections the node has. Degree equals the number of connections. The name of each node is an abbreviation, where an asterisk indicates multiple members of the TF family are included in the node (e.g., Rfx* for Rfx1, Rfx2, Rfx3, Rfx4) and when a node name is followed by + integer, as in “Mef2* + 1”, it indicates that there are TFs in the Mef2 family plus one other TF. All TFs associated with each node are listed in Additional file 1: Table S7. The number in parenthesis next to the node name indicatea the number of substage-negatively-concordant genes associated with the TF cluster. Figure S6. Expression of TF and its target genes for each substage. The difference of mean gene expression at 8 dpp of the TF and its target genes at each time point. To illustrate the overall pattern, a smoothed line was fit to the substage-specific gene expression. Color of lines represents the substage of target genes. Gene expression is shown as average log2(TPM + 1) across time point replicates. (ZIP 13261 kb)

本文件包含补充图S1至S6的微型网页，以及交互式表达绘图的访问链接。该微型网页亦可通过http://carterdev.jax.org/dtx/a1/index.html获取。

补充图S1：亚阶段一致性基因的表达。(A-H) 热图展示了各时间点下亚阶段一致性基因的基因表达情况，基因表达以对应时间点重复样本的平均log₂(TPM+1)值呈现。

补充图S2：亚阶段负一致性基因的表达。(A-H) 热图展示了各时间点下亚阶段负一致性基因的基因表达情况，基因表达以对应时间点重复样本的平均log₂(TPM+1)值呈现。

补充图S3：通过定量实时逆转录PCR(qRT-PCR)验证RNA-seq表达模式。上方箱线图展示了产后第10天(10 dpp)与产后第16天(16 dpp)时通过RNA-seq检测到的基因表达；下方柱状图展示了使用产后第10天与第16天的3只小鼠混合睾丸分离得到的生殖细胞，通过qRT-PCR检测到的基因表达。其中，RNA-seq的基因表达以log₂(TPM+1)值呈现，qRT-PCR的基因表达以相对于Actb基因的表达倍数变化值呈现。

补充图S4：转录因子(Transcription Factor, TF)弧图，展示一致性基因列表在各亚阶段的最显著转录因子。节点颜色代表所属亚阶段，节点大小与显著性水平正相关（节点越大代表显著性越高，所有节点的iRegulon标准化富集得分(Normalized Enrichment Score, NES)≥4，均具有高度统计学显著性）。弧的宽度对应两节点间共享的转录因子数量，弧越宽则共享转录因子越多。若某节点的转录因子集合为另一节点转录因子集合的子集，则两节点之间存在连接。弧的颜色（即节点度数）与节点的总连接数相关，度数即为该节点的连接总数。每个节点的名称为缩写形式，其中星号(*)表示该节点包含一个转录因子家族的多个成员（例如Rfx*代表Rfx1、Rfx2、Rfx3、Rfx4）；若节点名称后带有“+整数”格式（如“Mef2* +1”），则表示该节点包含Mef2家族的转录因子外加1个其他转录因子。每个节点对应的所有转录因子详见附加文件1的表S6。节点名称旁括号内的数字代表该转录因子簇关联的亚阶段一致性基因数量。

补充图S5：转录因子(Transcription Factor, TF)弧图，展示负一致性基因列表在各亚阶段的最显著转录因子。节点颜色代表所属亚阶段，节点大小与显著性水平正相关（节点越大代表显著性越高，所有节点的iRegulon NES≥4，均具有高度统计学显著性）。弧的宽度对应两节点间共享的转录因子数量，弧越宽则共享转录因子越多。若某节点的转录因子集合为另一节点转录因子集合的子集，则两节点之间存在连接。弧的颜色（即节点度数）与节点的总连接数相关，度数即为该节点的连接总数。每个节点的名称为缩写形式，其中星号(*)表示该节点包含一个转录因子家族的多个成员（例如Rfx*代表Rfx1、Rfx2、Rfx3、Rfx4）；若节点名称后带有“+整数”格式（如“Mef2* +1”），则表示该节点包含Mef2家族的转录因子外加1个其他转录因子。每个节点对应的所有转录因子详见附加文件1的表S7。节点名称旁括号内的数字代表该转录因子簇关联的亚阶段负一致性基因数量。

补充图S6：各亚阶段转录因子及其靶基因的表达情况。展示了各时间点下转录因子及其靶基因在产后第8天(8 dpp)时的平均基因表达差异。为直观呈现整体表达模式，我们对亚阶段特异性基因的表达数据拟合了平滑曲线。曲线颜色代表靶基因所属的亚阶段。基因表达以各时间点重复样本的平均log₂(TPM+1)值呈现。（ZIP格式，大小13261 kb）