Single-cell RNA-seq data of sorted bone marrow CD19+B220+ B cells from Cyldflx/flx mice and Mb1Cre-Cyldflx/flx mouse models

CYLD, a protein encoded by the Cyld gene, is a deubiquitinase involved in signalling events rather than proteasomal degradation. Mutations in CYLD are associated with familial cylindromatosis, leading to benign skin tumours. CYLD downregulates various solid and haematological tumours by negatively regulating signalling pathways like NF-κB, Wnt/β-catenin, and Notch. The most common Cyld splice variant encodes a 956 amino acid polypeptide with a catalytic domain at the carboxy-terminal, CAP-Gly domains at the N-terminal, and other domains in the central region. It interacts with various proteins, including tubulin, microtubules, and NEMO. CYLD is implicated in diverse functions such as cell proliferation, apoptosis, inflammation, spermatogenesis, and immune responses. Its role in B cell lymphopoiesis is less clear, with some studies showing increased B cell activation in Cyld-/- mice, while others suggest minimal impact on B cell maturation. To clarify CYLD's role in B cell lymphopoiesis, transgenic animals with B cell-specific catalytic inactivation of CYLD were created, revealing that CYLD plays a significant role in B cell maturation and function, with its disruption severely impeding B cell responses. Mb1Cre-Cyldflx/flx mice were generated by crossing a neo- derivative (Cyldfl9/fl9 neo-) of the previously developed Cyldfl9/fl9 mice25 with Mb1Cre mice33 (provided by Prof. M. Reth)34. In Cyldfl9/fl9 mice, loxP sites flank the 9th exon and when recombination occurs in the presence of Cre recombinase, the ensuing exons, including the gene region encoding for the catalytic domain, are out of frame and not expressed. All mice were maintained under specific pathogen-free (SPF) conditions at the animal laboratory facility of the IRCCS Ospedale San Raffaele, Milan, Italy. Mice of experimental groups were age-matched (3 and 6 months), of both genders and, in most cases, littermates. All animal experiments were approved by the Animal Ethics Committee of the IRCCS Ospedale San Raffaele for compliance to European regulations and licensed by the National Veterinary Administration authorities of Italy. For single-cell RNA sequencing, sorted bone marrow CD19+B220+ B cells were collected in RPMI medium (Euroclone, ECB2000) supplemented with 10% FBS (Euroclone, ECS0180L). Cell viability was then determined using Trypan Blue exclusion (Sigma-Aldrich, T8154-EA) and TC20 Automated Cell Counter (Bio-rad). 4000 cells were encapsulated using the Chromium Controller platform (10X Genomics) and libraries were prepared using the Chromium Next GEM Single Cell 3' Kit v3.1 (10X Genomics). Final libraries were sequenced according to the manufacturer's instructions on Novaseq6000.CYLD是由Cyld基因编码的蛋白质，属于去泛素化酶（deubiquitinase），其功能参与信号转导事件，而非蛋白酶体降解过程。CYLD的突变与家族性圆柱瘤病（familial cylindromatosis）相关，可引发良性皮肤肿瘤。CYLD通过负调控NF-κB、Wnt/β-连环蛋白（Wnt/β-catenin）和Notch等信号通路，对多种实体瘤和血液系统肿瘤发挥下调作用。最常见的Cyld剪接变体编码一条含956个氨基酸的多肽，其羧基端具有催化结构域，氨基端含有CAP-Gly结构域，中央区域则包含其他结构域。该蛋白可与微管蛋白（tubulin）、微管（microtubules）以及NEMO等多种蛋白质相互作用。CYLD参与多种生物学过程，包括细胞增殖、细胞凋亡、炎症反应、精子发生以及免疫应答。其在B细胞淋巴生成中的作用尚不明确：部分研究显示Cyld基因敲除（Cyld-/-）小鼠的B细胞激活增强，而另有研究表明其对B细胞成熟的影响极小。为阐明CYLD在B细胞淋巴生成中的作用，研究人员构建了B细胞特异性敲除CYLD催化活性的转基因动物，结果发现CYLD对B细胞成熟与功能具有重要调控作用，其功能缺失会严重损害B细胞应答。Mb1Cre-Cyldflx/flx小鼠的构建方式为：将此前开发的Cyldfl9/fl9小鼠的新霉素抗性基因衍生株（Cyldfl9/fl9 neo-）与Mb1Cre小鼠33（由M. Reth教授惠赠34）杂交获得。在Cyldfl9/fl9小鼠中，loxP位点位于第9号外显子两侧；当Cre重组酶存在时发生重组，后续的外显子（包括编码催化结构域的基因区域）会出现读码框移位，无法正常表达。所有小鼠均在意大利米兰IRCCS圣拉斐尔医院动物实验中心的无特定病原体（specific pathogen-free, SPF）环境中饲养。实验分组小鼠均为年龄匹配的3月龄和6月龄个体，涵盖两种性别，且多数为同窝仔鼠。所有动物实验均经IRCCS圣拉斐尔医院动物伦理委员会批准，符合欧洲法规要求，并获得意大利国家兽医管理部门的许可。 对于单细胞RNA测序（single-cell RNA sequencing, scRNA-seq），将分选得到的骨髓CD19+B220+ B细胞收集于添加了10%胎牛血清（fetal bovine serum, FBS）的RPMI培养基（Euroclone, ECB2000）中。随后采用台盼蓝排斥法（Trypan Blue exclusion, Sigma-Aldrich, T8154-EA）结合TC20全自动细胞计数仪（Bio-rad）测定细胞活力。使用Chromium Controller平台（10X Genomics）对4000个细胞进行包裹，再采用Chromium Next GEM Single Cell 3' Kit v3.1（10X Genomics）制备测序文库。最终文库按照制造商的操作指南在Novaseq6000测序平台上完成测序。