In the process of synaptic formation, neurons must not only adhere to specific principles when selecting synaptic partners but also possess mechanisms to avoid undesirable connections. Yet, the strategies employed to prevent unwarranted associations have remained largely unknown. In our study, we have identified the pivotal role of combinatorial clustered protocadherin gamma (γ-PCDH) expression in orchestrating synaptic connectivity in the mouse neocortex. Through 5-prime end single-cell sequencing, we unveiled the intricate combinatorial expression patterns of γ-PCDH variable isoforms within neocortical neurons. Furthermore, our whole-cell patch-clamp recordings demonstrated that as the similarity in this combinatorial pattern among neurons increased, their synaptic connectivity decreased. Our findings elucidate a sophisticated molecular mechanism governing the construction of neural networks in the mouse neocortex.

1, Single-cell dissociation. 2, FACS with DAPI.

3, Using Chromium Single Cell V(D)J Enrichment Kit, Mouse T Cell, PN-1000071 Kit (switching the primer with pcdhg specific primers) to get 5' end RNA seq.

4, Get clean reads from Novagene  Cellranger v3.0 for gene expression

5, Using  custom pipeline (written in the Matlab code)and Bowtie for pcdhg 5'end;