小柯机器人

美国威尔康奈尔医学院Hagen U. Tilgner小组取得一项新突破。他们的最新研究利用单核异构体RNA测序解锁冷冻脑组织中的条形码外显子连接。相关论文发表在2022年3月7日出版的《自然-生物技术》杂志上。

研究人员研发了单核异构体RNA测序(SnISOr-Seq)。使用微流体、基于PCR的伪影去除、目标富集和长读长测序，与原始长读长单核测序相比，SnISOr-Seq将条形码、跨越外显子的长读长精度提高了7.5倍。研究人员对成人额叶皮层进行SnISOr-Seq，发现与自闭症相关的外显子表现出协调性和高度细胞类型特异性包含。

研究发现了两种不同的组合模式：独特的神经细胞类型，富含TSS外显子、外显子多腺苷酸化位点和非相邻外显子对，以及在一种细胞类型中具有多种配置、富含相邻外显子对的神经细胞类型。

最后，研究人员发现人类特异性外显子几乎与保守外显子一样紧密协调，这意味着在进化过程中可以迅速建立协调。SnISOr-Seq可在人脑和任何冷冻或难以分离的样本中进行细胞类型特异性长读长异构体分析。

据了解，单核RNA测序可在基因水平上表征细胞类型。然而，与单细胞测序相比，许多单核cDNA是纯内含子，缺乏条形码，这阻碍了异构体的研究。

附：英文原文

Title: Single-nuclei isoform RNA sequencing unlocks barcoded exon connectivity in frozen brain tissue

Author: Hardwick, Simon A., Hu, Wen, Joglekar, Anoushka, Fan, Li, Collier, Paul G., Foord, Careen, Balacco, Jennifer, Lanjewar, Samantha, Sampson, Maureen McGuirk, Koopmans, Frank, Prjibelski, Andrey D., Mikheenko, Alla, Belchikov, Natan, Jarroux, Julien, Lucas, Anne Bergstrom, Palkovits, Mikls, Luo, Wenjie, Milner, Teresa A., Ndhlovu, Lishomwa C., Smit, August B., Trojanowski, John Q., Lee, Virginia M. Y., Fedrigo, Olivier, Sloan, Steven A., Tombcz, Dra, Ross, M. Elizabeth, Jarvis, Erich, Boldogki, Zsolt, Gan, Li, Tilgner, Hagen U.

Issue&Volume: 2022-03-07

Abstract: Single-nuclei RNA sequencing characterizes cell types at the gene level. However, compared to single-cell approaches, many single-nuclei cDNAs are purely intronic, lack barcodes and hinder the study of isoforms. Here we present single-nuclei isoform RNA sequencing (SnISOr-Seq). Using microfluidics, PCR-based artifact removal, target enrichment and long-read sequencing, SnISOr-Seq increased barcoded, exon-spanning long reads 7.5-fold compared to naive long-read single-nuclei sequencing. We applied SnISOr-Seq to adult human frontal cortex and found that exons associated with autism exhibit coordinated and highly cell-type-specific inclusion. We found two distinct combination patterns: those distinguishing neural cell types, enriched in TSS-exon, exon-polyadenylation-site and non-adjacent exon pairs, and those with multiple configurations within one cell type, enriched in adjacent exon pairs. Finally, we observed that human-specific exons are almost as tightly coordinated as conserved exons, implying that coordination can be rapidly established during evolution. SnISOr-Seq enables cell-type-specific long-read isoform analysis in human brain and in any frozen or hard-to-dissociate sample. Complete RNA isoforms are captured by a new single-nuclei sequencing method.

DOI: 10.1038/s41587-022-01231-3

Source: https://www.nature.com/articles/s41587-022-01231-3

Nature Biotechnology：《自然—生物技术》，创刊于1996年。隶属于施普林格·自然出版集团，最新IF：68.164
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