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. 

The pertinent code can be found here: https://github.com/BiodataAnalysisGroup/Cyld-regulation-of-bcell-maturation