Spatial Transcriptomics Using Multiplexed Deterministic Barcoding in Tissue

In this study, we present a multiplexed version of Deterministic Barcoding in Tissue (xDBiT) to acquire spatially resolved transcriptomes of nine tissue sections in parallel. New microfluidic chips were developed to spatially encode mRNAs over a total tissue area of 1.17 cm2 with spots of 50 m50 m. Optimization of the biochemical protocol increased read and gene counts per spot by one order of magnitude compared with previous reports. Furthermore, the introduction of alignment markers allows seamless registration of images and spatial transcriptomic spot coordinates. Together with technological advances, we provide an open-source computational pipeline to transform raw sequencing data from xDBiT experiments into the AnnData format. The functionality of xDBiT was demonstrated by the acquisition of 16 spatially resolved transcriptomic datasets from five different murine organs, including cerebellum, liver, kidney, spleen, and heart. Factor analysis and deconvolution of xDBiT spatial transcriptomes allowed for in-depth characterization of the murine kidney. Spatial transcriptomics samples of cerebellum, heart, liver, spleen and kidney from 3-month old mice.本研究提出了组织确定性条形码技术（Deterministic Barcoding in Tissue，DBiT）的多重化版本xDBiT，可并行获取9个组织切片的空间分辨转录组。我们研发了新型微流控芯片，可在总面积达1.17 cm²的组织区域上，以50 μm×50 μm的斑点实现mRNA的空间编码。对生化实验流程的优化，使每个斑点的读取计数与基因计数较既往研究提升了一个数量级。此外，引入对齐标记可实现图像与空间转录组斑点坐标的无缝配准。结合上述技术进展，我们开源了一套可将xDBiT实验产生的原始测序数据转换为AnnData格式的计算流程。我们通过从5种不同小鼠器官（包括小脑、肝脏、肾脏、脾脏及心脏）中获取的16套空间分辨转录组数据集，验证了xDBiT的功能有效性。对xDBiT获取的空间转录组进行因子分析与反卷积，可实现小鼠肾脏的深度表征。本研究的样本均为3月龄小鼠的小脑、心脏、肝脏、脾脏与肾脏的空间转录组样本。