Transcription profiling by high throughput sequencing of purified Sertoli cells, spermatogonia, spermatocytes, spermatids and spermatozoa from mice

Understanding the extent of genomic transcription and its functional relevance is a central goal in genomics research. However, detailed genome-wide investigations of transcriptome complexities in major mammalian organs and their underlying cellular sources, transcriptional mechanisms, and functional relevance have been scarce. Here we first show, using extensive RNA-seq data, that transcription of both functional and nonfunctional genomic elements is substantially more widespread in the testis than in other organs across representative mammals. By scrutinizing the transcriptomes of all main testicular cell types in the mouse, we then reveal that meiotic spermatocytes and especially post-meiotic round spermatids have remarkably diverse transcriptomes, which explains the high transcriptome complexity of the testis as a whole. The widespread transcriptional activity in spermatocytes and spermatids encompasses protein-coding genes and long noncoding RNA genes but also poorly conserved intergenic sequences, suggesting that much of it is not of immediate functional relevance. Rather, our analyses of genome-wide epigenetic data show that this prevalent transcription, which apparently promoted the birth of new genes during evolution, results from a highly permissive chromatin state during and after meiosis that may ultimately facilitate the replacement of histones by protamines during late spermatogenesis. To study the cellular source and mechanisms of high transcriptome complexity in the mammalian testis, we generated strand-specific deep coverage RNA-Seq data for purified sertoli cells, spermatogonia, spermatocytes, spermatids and spermatozoa as well as for brain, liver and the whole testis from the mouse. We prepared 8 sequencing libraries for the polyadenylated RNA fraction of each sample and sequenced each library in 3 lanes of the Illumina Genome Analyser IIx platform, yielding a total of >60 millions strand-specific reads of 76 base pairs per sample. In addition, we generated ChIP-Seq data for the H3K4me2 modification as well as RRBS data for brain, liver, testis, spermatocytes and spermatids. RNA-seq, ChIP-seq and RRBS data were generated from the same individual or pool of individuals, in the case of purified cells. RNA-Seq data for purified sertoli cells, spermatogonia, spermatocytes, spermatids and spermatozoa解析基因组转录的范围及其功能相关性，是基因组学研究的核心目标之一。然而，针对主要哺乳动物器官的转录组复杂性及其潜在细胞来源、转录调控机制与功能相关性的全基因组精细研究仍较为匮乏。本研究首先借助大规模RNA测序（RNA-seq）数据证实：在代表性哺乳动物中，功能性与非功能性基因组元件的转录行为在睾丸中均远较其他器官更为广泛。随后，通过解析小鼠体内所有主要睾丸细胞类型的转录组，本研究揭示：减数分裂精母细胞（spermatocytes），尤其是减数分裂后圆形精子细胞（round spermatids），拥有极为多样的转录组，这正是整体睾丸转录组复杂性较高的原因。精母细胞与精子细胞中广泛存在的转录活性，既涵盖蛋白编码基因与长链非编码RNA（long noncoding RNA）基因，也包含保守性较低的基因间序列，这表明其中多数转录事件并不具备直接的功能相关性。进一步的全基因组表观组数据分析显示，这类普遍存在的转录事件（其在演化过程中曾推动新基因的诞生），源于减数分裂期间及之后的高度开放染色质状态；该状态最终可促进晚期精子发生过程中组蛋白被鱼精蛋白所替换。 为探究哺乳动物睾丸高转录组复杂性的细胞来源与调控机制，本研究针对小鼠的纯化支持细胞（Sertoli cells）、精原细胞（spermatogonia）、精母细胞（spermatocytes）、精子细胞（spermatids）、精子（spermatozoa），以及大脑、肝脏与完整睾丸，构建了链特异性深度覆盖RNA-seq数据。我们为每份样本的聚腺苷酸化RNA组分构建了8个测序文库，并将每份文库在伊卢米纳基因组分析仪IIx（Illumina Genome Analyser IIx）平台的3个泳道中进行测序，最终每份样本可获得超过6000万条76碱基对的链特异性测序读段。此外，我们还针对大脑、肝脏、睾丸、精母细胞与精子细胞，生成了组蛋白H3赖氨酸4二甲基化修饰（H3K4me2）的染色质免疫共沉淀测序（ChIP-seq）数据，以及简化代表性亚硫酸氢盐测序（RRBS）数据。对于纯化细胞样本，RNA-seq、ChIP-seq与RRBS数据均来自同一实验个体或混合样本。针对纯化支持细胞（Sertoli cells）、精原细胞（spermatogonia）、精母细胞（spermatocytes）、精子细胞（spermatids）与精子（spermatozoa）的RNA-seq数据