An optimized protocol for retina single-cell RNA sequencing [snRNA-Seq]

In this work, we compared different protocols to prepare single-cell suspensions used for scRNAseq and suggest an optimized dissociation protocol for mouse retina, which preserves cell morphology to a higher level leading to an overall increase of gene number per cell. We compared scRNAseq libraries generated with our optimized protocol to publicly available scRNAseq data of mouse retina. We further demonstrate a pipeline to reduce noise in scRNAseq caused by multiplets and ambient RNA. Mouse retina were dissected and dissociated using different protocols. Libraries were generated using 10x Genomics' "Chromium Single Cell Gene Expression" system. We compared cell suspensions, gene number per cell, level of ambient RNA and the overall gene detection rate within across cell types and protocols.本研究针对用于单细胞RNA测序（single-cell RNA sequencing，scRNAseq）的单细胞悬液制备方案开展系统对比分析，并提出一种针对小鼠视网膜的优化解离方案。该方案可更高程度保留细胞形态，进而整体提升单个细胞的检测基因数。我们将采用本优化方案构建的scRNAseq文库与公开可用的小鼠视网膜scRNAseq数据集进行比对。此外，我们还展示了一套可有效降低由细胞多重体与游离RNA（ambient RNA）所引发的scRNAseq测序噪声的分析流程。本研究采用多种不同方案对小鼠视网膜进行解剖与解离，文库构建均使用10x Genomics公司的"Chromium单细胞基因表达"系统。我们从细胞悬液状态、单个细胞的检测基因数、游离RNA水平，以及不同细胞类型和各实验方案下的整体基因检出率等多个维度开展了比对分析。