概要: Angiosperms possess a characteristic life cycle with an alternation of sporophyte and gametophyte generations, which happens in plant organs like pistils. Rice pistils contain ovules and receive pollen for successful fertilization to produce grains. The cellular expression profile in rice pistils is largely unknown. Here we show a cell atlas of rice pistils before fertilization by droplet-based single-nucleus RNA sequencing. The Ab initio marker identification validated by in situ hybridization assists the cell-type annotation, revealing cell heterogeneity between ovule and carpel lineages.
项目整体设计: Perform snRNA-seq for 1N (haploid; gametophyte) and 2N (diploid; sporophyte) nuclei and bulk RNA-seq for nuclei of pistils and whole cells of upper and lower part of pistils
Rice (Oryza sativa ssp. Japonica cultivar Nipponbare) plants were grown in the potting soil mixed with sand in climate chambers with a relative humidity of 70%, under the following circadian rhythm: 10 h light at 30℃ and 14 h dark at 25℃.
处理方案:
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提取方案:
Dissected pistils were frozen in liquid nitrogen and ground to powder. RNeasy Micro Kit (Qiagen) was used for RNA extraction. For bulk nuclei RNA sequencing, the powder was suspended by nucleus isolation buffer. Then, the crude nuclei were enriched from the slurry by applying cell strainer, and were subsequently purified by PI-labeled FANS. The purified nuclei were subjected to RNA extraction using RNeasy Micro Kit (Qiagen).
建库方案:
For RNA-seq, the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB) was used to construct the library. For bulk nuclei RNA sequencing, RNA libraries for RNA-seq were prepared using SMART-Seq Library Prep Kit following manufacturer's protocols.
