Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, CA, USA.
Wu Tsai Neurosciences Institute, Stanford University School of Medicine, Stanford, CA, USA.
Nucleic Acids Res. 2021 Jan 25;49(2):e11. doi: 10.1093/nar/gkaa1127.
Massively-parallel single-cell and single-nucleus RNA sequencing (scRNA-seq, snRNA-seq) requires extensive sequencing to achieve proper per-cell coverage, making sequencing resources and availability of sequencers critical factors for conducting deep transcriptional profiling. CoolMPS is a novel sequencing-by-synthesis approach that relies on nucleotide labeling by re-usable antibodies, but whether it is applicable to snRNA-seq has not been tested. Here, we use a low-cost and off-the-shelf protocol to chemically convert libraries generated with the widely-used Chromium 10X technology to be sequenceable with CoolMPS technology. To assess the quality and performance of converted libraries sequenced with CoolMPS, we generated a snRNA-seq dataset from the hippocampus of young and old mice. Native libraries were sequenced on an Illumina Novaseq and libraries that were converted to be compatible with CoolMPS were sequenced on a DNBSEQ-400RS. CoolMPS-derived data faithfully replicated key characteristics of the native library dataset, including correct estimation of ambient RNA-contamination, detection of captured cells, cell clustering results, spatial marker gene expression, inter- and intra-replicate differences and gene expression changes during aging. In conclusion, our results show that CoolMPS provides a viable alternative to standard sequencing of RNA from droplet-based libraries.
大规模平行单细胞和单细胞核 RNA 测序（scRNA-seq，snRNA-seq）需要进行大量测序才能实现适当的每个细胞覆盖度，因此测序资源和测序仪的可用性成为进行深度转录组分析的关键因素。CoolMPS 是一种新的测序合成方法，它依赖于可重复使用的抗体进行核苷酸标记，但它是否适用于 snRNA-seq 尚未得到验证。在这里，我们使用一种低成本且现成的方案，将广泛使用的 10X Chromium 技术生成的文库进行化学转化，使其能够与 CoolMPS 技术兼容。为了评估使用 CoolMPS 测序的转化文库的质量和性能，我们从小鼠的海马体中生成了一个 snRNA-seq 数据集。原始文库在 Illumina Novaseq 上进行测序，而转化为与 CoolMPS 兼容的文库则在 DNBSEQ-400RS 上进行测序。CoolMPS 衍生的数据忠实地复制了原始文库数据集的关键特征，包括对环境 RNA 污染的正确估计、捕获细胞的检测、细胞聚类结果、空间标记基因表达、内和间重复差异以及衰老过程中的基因表达变化。总之，我们的结果表明，CoolMPS 为基于液滴的文库的 RNA 标准测序提供了一种可行的替代方案。
