HyPR-seq: Single-cell quantification of chosen RNAs via hybridization and sequencing of DNA probes (scRNA-seq dataset)

Single-cell quantification of RNAs is important for understanding cellular heterogeneity and gene regulation, yet current approaches suffer from low sensitivity for individual transcripts, limiting their utility for many applications. Here we present HyPR-seq, a method to sensitively quantify the expression of 10 to 100 chosen genes in single cells. HyPR-seq involves hybridizing DNA probes to RNA, distributing cells into nanoliter droplets, amplifying the probes with PCR, and sequencing the amplicons to quantify the expression of chosen genes. HyPR-seq achieves high sensitivity for individual transcripts, detects non-polyadenylated and low-abundance transcripts, and can profile up to 100,000 single cells. We demonstrate how HyPR-seq can profile the effects of CRISPR perturbations in pooled screens, detect time-resolved changes in gene expression via measurements of gene introns, and detect rare transcripts and quantify cell type frequencies in tissue using low-abundance marker genes. By sensitively quantifying individual transcripts and directing sequencing power to genes of interest, HyPR-seq reduces costs by up to 100-fold compared to whole-transcriptome scRNA-seq approaches for these applications. HyPR-seq will be a powerful method for targeted RNA profiling in single cells. We performed HyPR-seq in K562 cells (45 samples), THP1 cells (8 samples), and cells from wild-type and diabetic murine kidney (4 samples). HyPR-seq data from K562 cells includes 2 samples with probes targeting highly expressed genes and some introns, 1 sample representing a single-cell mixing experiment, and 42 samples representing a timecourse study to look at the kinetics of intron expression after promoter inhibition with CRISPR interference (3 samples each at 14 timepoints). THP1 samples include 4 replicates with LPS stimulation and 4 samples without. We also performed RNA sequencing on THP1 cells with and without LPS (3 replicates each) using the Smart-seq2 protocol in bulk. Kidney cells were obtained from 12-week old BTBR mice, 2 wt/wt and 2 ob/ob.RNA的单细胞定量对于解析细胞异质性与基因调控机制具有重要意义，但现有方法对单个转录本的检测灵敏度较低，限制了其在诸多应用场景中的实用价值。本研究提出HyPR-seq技术——一种可在单细胞中对10至100个选定基因的表达水平进行高灵敏定量的实验方法。该技术的实验流程包括：将DNA探针与RNA进行杂交，将细胞分配至纳升液滴中，通过聚合酶链式反应（PCR, polymerase chain reaction）扩增探针，随后对扩增产物进行测序以定量选定基因的表达水平。HyPR-seq对单个转录本具有较高检测灵敏度，可检测非多聚腺苷酸化（non-polyadenylated）及低丰度转录本，且最高可对10万个单细胞开展表达谱分析。本研究验证了HyPR-seq可用于混合筛选中成簇规律间隔短回文重复序列（CRISPR, clustered regularly interspaced short palindromic repeats）扰动效应的表达谱分析，通过检测基因内含子（intron）序列实现基因表达的时间动态变化监测，还可借助低丰度标记基因（marker gene）检测组织中的稀有转录本并定量细胞类型占比。通过对单个转录本进行高灵敏定量并将测序通量聚焦于目标基因，HyPR-seq相较于全转录组单细胞RNA测序（scRNA-seq, single-cell RNA sequencing）技术，在这类应用中可将成本降低最高达100倍。HyPR-seq将成为单细胞靶向RNA表达谱分析的高效技术工具。本研究分别在K562细胞（45个样本）、THP1细胞（8个样本）以及野生型与糖尿病模型小鼠肾脏细胞（4个样本）中开展了HyPR-seq实验。K562细胞的HyPR-seq数据集包含：2个使用靶向高表达基因及部分内含子探针的样本、1个单细胞混合实验样本，以及42个用于时间进程研究的样本——该研究旨在探究CRISPR干扰（CRISPR interference, CRISPRi）抑制启动子后内含子表达的动力学特征，14个时间点每个时间点设置3个生物学重复。THP1细胞样本包含4个脂多糖（LPS, lipopolysaccharide）刺激组样本与4个未刺激组样本。本研究同时采用Smart-seq2技术对有/无LPS刺激的THP1细胞进行了批量RNA测序（bulk RNA sequencing），每组设置3个生物学重复。肾脏细胞取自12周龄的BTBR小鼠，其中野生型纯合（wt/wt）与肥胖型纯合（ob/ob）小鼠各2只。