{"database":"NODE","file_versions":[],"scores":null,"additional":{"omics_type":["Single Cell Transcriptomics"],"submitter":["Di Wu Zhao"],"technology_type":["RNA-Seq"],"experiment_platform":["Illumina NovaSeq 6000"],"full_dataset_link":["https://www.biosino.org/node/experiment/detail/OEX00003668"],"experiment_library_layout":["Paired"],"experiment_library_selection":["RANDOM PCR"],"sample_count":["2"],"tissue":["['cell Line']"],"taxonomy":["['Mus musculus']"],"experiment_protocol":["Peripheral lymph nodes were harvested from mice with indicated genotypes, and grinded into single cells, followed by treatment with Dynabeads™ Untouched™ Mouse CD4 Cells Kit (Invitrogen, 11415D) to isolate the CD4+ T cells. The FACS sorting (SONY Cell Sorter, SH800S) was used to isolate the CD4+YFP+ Treg cells with purities > 99%. To generate enough materials, the lymph nodes from 3 Foxp3cre/wt or 6 Foxp3cre/wt;Rbx1fl/fl mice at age of 10 weeks were pooled and subjected to single-cell RNA sequencing by Sinotech Genimics Co., Ltd. (Shanghai, China). The single-cell capture was achieved by random distribution of a single-cell suspension across > 200,000 microwells through a limited dilution approach, and the wt and Rbx1-deficient Treg cells were distributed on two individual microwell plates. Beads with oligonucleotide barcodes were added to saturation so that a bead was paired with a cell in a microwell. Cell-lysis buffer was added to facilitate poly-adenylated RNA molecules hybridized to the beads. Beads were collected into a single tube for reverse transcription. Upon cDNA synthesis, each cDNA molecule was tagged on the 5’ end (that is, the 3’ end of a mRNA transcript) with a molecular index and cell label to indicate its cell of origin. Whole transcriptome libraries were prepared using the BD Resolve system for single-cell whole-transcriptome amplification workflow (BD Genomics) to capture transcriptomic information of the sorted single cells. Briefly, the second strand cDNA was synthesized, followed by ligation of the adaptor for universal amplification. Eighteen cycles of PCR were used to amplify the adaptor-ligated cDNA products. Sequencing libraries were prepared using random priming PCR of the whole-transcriptome amplification products to enrich the 3’ end of the transcripts linked with the cell labels and molecular indices. Sequencing libraries were quantified using a High Sensitivity DNA chip (Agilent) on a Bioanalyzer 2100 and the Qubit High Sensitivity DNA assay (Thermo Fisher Scientific). Approximate 1.5pM of the library for each sample was loaded onto a NextSeq 500 system and sequenced using High Output sequencing kits (75× 2bp) (Illumina). After discarding the cells with high mitochondrial gene, 10,048 and 9,736 cells were identified for wt and Rbx1-deficient Treg cells, with an average number of reads of 23,321.27 vs. 22202.55, and 1,080.16 vs. 870.28 of genes detected per cell, respectively."],"repository":["NODE"],"additional_accession":[]},"is_claimable":false,"name":"JZ201912111353_deep","description":"Cullin-RING Ligases (CRLs), upon activation by neddylation, play the key roles in regulating many biological processes. However, how CRL-neddylation regulates the function of Treg cells remains elusive. Here we show that mice with Treg-specific deletion of Rbx1, a RING component of CRLs required for its activity, developed an early-onset fetal inflammatory disorders and death at day ~25 after birth with disrupted homeostasis and impaired suppressive functions of Treg cells. Specifically, Rbx1 is essential for maintenance of the effector subpopulations in Treg cells, and regulates several inflammatory pathways. Similar phenotypes were seen in mice with deletion of Ube2m, a neddylation E2, in Treg cells, but with much lesser severity. Interestingly, Treg-specific deletion of Rbx2/Sag or Ube2f, the family member of Rbx1 or Ube2m, respectively, had no obvious phenotype. Thus, the Ube2m-Rbx1 axis is required for the maintenance of homeostasis and functions of Treg cells; and Rbx1 has Ube2m-independent roles in the fitness of Treg cells, suggesting a layer of complexity in neddylation activation of CRLs.","dates":{"publication":"2022-01-04","submission":"2020-05-26"},"accession":"OEX00003668","cross_references":{"NODE":["OEP00000926"]}}