Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
Cardiovascular Division, Brigham and Women's Hospital, Boston, Massachusetts, USA.
Curr Protoc. 2022 Oct;2(10):e579. doi: 10.1002/cpz1.579.
This protocol describes a robust pipeline for simultaneously analyzing multiple samples by single-nucleus (sn)RNA-seq. cDNA obtained from each single sample are labeled with the same lipid-coupled oligonucleotide barcode (10X Genomics). Nuclei from as many as 12 individual samples can be pooled together and simultaneously processed for cDNA library construction and subsequent DNA sequencing. While previous protocols using lipid-coupled oligonucleotide barcodes were optimized for analysis of samples consisting of viable cells, this protocol is optimized for analyses of quick-frozen cell samples. The protocol ensures efficient recovery of nuclei both by incorporating high sucrose buffered solutions and by including a tracking dye (trypan blue) during nuclei isolation. The protocol also describes a procedure for removing single nuclei 'artifacts' by removing cell debris prior to single nuclear fractionation. This protocol informs the use of computational tools for filtering poorly labeled nuclei and assigning sample identity using barcode unique molecular identifier (UMI) read counts percentages. The computational pipeline is applicable to either cultured or primary, fresh or frozen cells, regardless of their cell types and species. Overall, this protocol reduces batch effects and experimental costs while enhancing sample comparison. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Labeling cells with lipid oligo barcodes and generating multiplexed single-nucleus RNA-seq libraries Basic Protocol 2: Bioinformatic deconvolution of the multiplexed snRNAseq libraries.
本方案描述了一种通过单细胞 RNA-seq 同时分析多个样本的稳健流程。从每个单个样本获得的 cDNA 用相同的脂质偶联寡核苷酸条形码（10X Genomics）标记。多达 12 个个体样本的核可以混合在一起，同时进行 cDNA 文库构建和随后的 DNA 测序。虽然之前使用脂质偶联寡核苷酸条形码的方案是针对由活细胞组成的样本进行优化的，但该方案是针对快速冷冻细胞样本的分析进行优化的。该方案通过包含高蔗糖缓冲溶液并在核分离过程中包含跟踪染料（台盼蓝）来确保核的有效回收。该方案还描述了一种通过在单细胞分离之前去除细胞碎片来去除单个核“假象”的程序。该方案告知使用计算工具过滤标记不良的核，并使用条形码唯一分子标识符 (UMI) 读数百分比分配样本身份。该计算流程适用于培养的或原代的、新鲜的或冷冻的细胞，无论其细胞类型和物种如何。总体而言，该方案减少了批次效应和实验成本，同时增强了样本比较。© 2022 威利父子公司。基本方案 1：用脂质寡核苷酸条形码标记细胞并生成多路复用的单细胞 RNA-seq 文库 基本方案 2：多路复用 snRNAseq 文库的生物信息学反卷积。
