Single-nuclei isoform RNA sequencing reveals combination patterns of transcript elements across human brain cell types

Single-nuclei RNA-Seq is widely employed to investigate cell types, especially of human brain and frozen samples. In contrast to single-cell approaches, many single-nuclei reads are purely intronic. Here, using microfluidics, PCR-based artifact removal, target enrichment, and long-read sequencing, we developed single-nuclei isoform RNA-sequencing ('SnISOr-Seq'), and applied it to human adult frontal cortex. SnISOr-Seq dramatically increased the fraction of informative reads. We found that exons associated with autism exhibit coordinated and highly cell-type specific inclusion. We discovered two distinct combination patterns: first, those distinguishing neural cell types, enriched in TSS-exon, exon-polyA-site, and non-adjacent exon pairs. Second, those with multiple configurations within one cell type, enriched in adjacent exon pairs. Furthermore, adjacent exons are predominantly mutually-associated, while distant exons are frequently mutually-exclusive. Finally, we observed that human-specific exons are almost as tightly coordinated as conserved exons. SnISOr-Seq enables single-nuclei long-read isoform analysis in human brain, and in any frozen or hard-to-dissociate sample. 10X single-nuclear RNA sequencing done on two replicates of human Frontal Cortex samples. PacBio done on the same cDNA, after linear PCR. 10X single-cell RNA sequencing followed by Oxford Nanopore long read sequencing as a control sample. FACS sorted nuclei obtained from Human prefrontal cortex samples for bulk RNA sequencing on Illumina, also used as control.单细胞核RNA测序（single-nuclei RNA-Seq）已被广泛应用于细胞类型鉴定领域，尤其适用于人类脑组织及冷冻样本。与单细胞测序策略相比，单细胞核测序产生的大量测序读段完全为内含子源性序列。本研究结合微流控技术、基于PCR的测序伪影去除、靶标富集及长读长测序技术，开发了单细胞核异构体RNA测序方法（single-nuclei isoform RNA-sequencing，简称SnISOr-Seq），并将其应用于人类成人前额叶皮层。SnISOr-Seq显著提升了有效测序读段的占比。研究发现，与自闭症相关的外显子呈现出协调一致且高度细胞类型特异性的剪接保留模式。本研究识别出两类截然不同的组合模式：其一为区分神经细胞类型的模式，这类模式富集于转录起始位点-外显子（TSS-exon）、外显子-多聚腺苷酸化位点（exon-polyA-site）及非相邻外显子对中；其二为单一种类细胞内存在多种可变构型的模式，这类模式富集于相邻外显子对中。此外，相邻外显子大多呈共关联状态，而远距离外显子则通常呈现互斥状态。最后，研究观察到人类特异性外显子的协调程度几乎与保守外显子相当。SnISOr-Seq可实现人类脑组织及所有冷冻样本或难解离样本的单细胞核长读长异构体分析。对两份重复的人类前额叶皮层样本开展10X单细胞核RNA测序；采用线性PCR扩增后的同一cDNA样本进行PacBio测序；以10X单细胞RNA测序结合牛津纳米孔长读长测序作为对照样本；从人类前额叶皮层样本中通过荧光激活细胞分选（Fluorescence-Activated Cell Sorting, FACS）分离得到的细胞核，用于Illumina平台的批量RNA测序，同样作为对照样本。