sRNA profiling during Oxytricha conjugation. Oxytricha trifallax

In Oxytricha, the somatic genome is responsible for vegetative growth, while the germline contributes DNA to the next sexual generation. Somatic nuclear development eliminates all transposons and other so-called "junk DNA", which constitute ~95% of the germline. We demonstrate that Piwi-interacting small RNAs (piRNAs) from the maternal nucleus can specify genomic regions for retention in this process. Oxytricha piRNAs map primarily to the somatic genome, representing the ~5% of the germline that is retained. Furthermore, injection of synthetic piRNAs corresponding to normally-deleted regions leads to their retention in subsequent generations. Our findings highlight small RNAs (sRNAs) as powerful transgenerational carriers of epigenetic information for genome programming. The backcross study here shows that the mating between an IES+ strain with the wild-type stain produces corresponding IES-containing sRNAs at 19 hr, and we provided the mapping to and the sequences of the specific loci of interest in the submission. As a control, wild-type cells do not produce such IES-containing sRNAs, and this analysis can be pulled out from the GSE35018 study since we provided mapping to the whole genome. The purpose of the 20 hr total sRNA sequencing study here is to show that the class of 27 nt sRNA is the major species of total sRNAs in Oxytricha at 20 hr, which we sequenced previously from Otiwi1-associated sRNAs at 12, 19, 23, and 30 hr (GSE35018). In addition, there is a less abundant class of small RNAs of 21-22 nt. These two classes are obvious by simply plotting the length distribution of the sRNA sequences. Overall design: We sequenced sRNAs from Contig22226.0 IES1+ strain backcrossed to wild-type parental strain at 19hr post-mixing, and found corresponding IES-containing sRNAs. As a control, wild-type cells do not produce such IES-containing sRNAs (see GSE35018). Total RNA from the backcrossing at 19hr were isolated with mirVana small RNA extraction kit (Ambion), and directly used for making Illumina sRNA libraries. Oxytricha total small RNA (sRNA) sequencing at 20 hr post conjugation shows that a class of 27 nt, 5'-U sRNAs dominates the sRNA population at 20 hr, and this class of sRNAs associate with Otiwi1 (see GSE35018 for Otiwi1-interacting sRNAs in Oxytricha). In addition, a much less abundant class of 21-22 nt sRNAs is present according to the length distribution.

在尖毛虫属（Oxytricha）中，体细胞基因组负责营养生长，而生殖系则为下一代有性生殖提供DNA。体细胞核发育过程会清除所有转座子及其他所谓的‘垃圾DNA’，这些序列约占生殖系基因组的95%。本研究证实，来自母细胞核的Piwi相互作用小RNA（Piwi-interacting small RNAs，piRNAs）可在该过程中指定需要保留的基因组区域。尖毛虫的piRNAs主要比对至体细胞基因组，对应生殖系基因组中约5%的保留序列。此外，向细胞注射对应正常被清除区域的合成piRNAs，可使这些区域在后续世代中得以保留。本研究结果表明，小RNA（small RNAs，sRNAs）是表观遗传信息跨代传递的有效载体，可介导基因组编程。本次回交实验显示，将携带内部消除序列（IES）的菌株与野生型菌株交配后，可在19小时检测到含IES的sRNAs，本提交材料中已提供对应目标特定位点的比对结果与序列。作为对照，野生型细胞不会产生此类含IES的sRNAs，该分析可从GSE35018研究中提取，因为我们已提供全基因组比对数据。本次20小时总sRNA测序实验的目的在于证实，27 nt长度的sRNA是尖毛虫在20小时时的主要总sRNA类群——我们此前曾在12、19、23和30小时从Otiwi1结合的sRNAs中检测到该类群（GSE35018）。此外，还存在一类丰度较低的21-22 nt小RNA。通过绘制sRNA序列的长度分布即可清晰区分这两类sRNA。实验设计：我们对混合后19小时的、经Contig22226.0 IES1+菌株与野生型亲本菌株回交得到的样本进行sRNA测序，检测到了含IES的sRNAs。作为对照，野生型细胞不会产生此类含IES的sRNAs（详见GSE35018）。回交样本在19小时的总RNA采用mirVana小RNA提取试剂盒（Ambion公司）分离，并直接用于构建Illumina小RNA文库。尖毛虫接合生殖后20小时的总小RNA（sRNAs）测序结果显示，一类27 nt、5'-U的sRNAs构成了该时间点的主要sRNA类群，且此类sRNAs可与Otiwi1结合（尖毛虫Otiwi1结合sRNAs的相关数据详见GSE35018）。此外，根据长度分布分析，还存在一类丰度显著更低的21-22 nt sRNAs。