Processed data objects for snPATHO-seq paper

Formalin-fixed paraffin-embedded (FFPE) samples are valuable but underutilized in single-cell omics research due to their low DNA and RNA quality. In this study, leveraging a recent advance in single-cell genomic technology, we introduce snPATHO-seq, a versatile method to derive high-quality single-nucleus transcriptomic data from FFPE samples. We benchmarked the performance of the snPATHO-seq workflow against existing 10x 3’ and Flex assays designed for frozen or fresh samples and highlighted the consistency in snRNA-seq data produced by all workflows. The snPATHO-seq workflow also demonstrated high robustness when tested across a wide range of normal and diseased FFPE tissue samples. When combined with FFPE spatial transcriptomic technologies such as FFPE Visium, the snPATHO-seq provides a multi-modal sampling approach for FFPE samples, allowing more comprehensive transcriptomic characterization.

Methods
Frozen PBMC samples were thawed and processed directly for 10x 3' and Flex chemistry.

For frozen breast cancer tissue samples, samples were dissociated into single-nucleus suspension before being processed for 10x 3' and Flex chemistry for gene expression analysis.

For FFPE tissue samples, nuclei were extracted using either the snPATHO-seq protocol or the 10x scFFPE protocol followed by 10x Flex chemistry processing for gene expression detection.

FFPE Visium data was generated using the 10x FFPE Visium CytAssist workflow according to the manufacturer's recommendation.

甲醛固定石蜡包埋（Formalin-fixed paraffin-embedded, FFPE）样本在单细胞组学研究中虽价值不菲却未得到充分利用，究其根源在于其DNA与RNA质量普遍偏低。本研究依托近期单细胞基因组学技术的突破性进展，推出了snPATHO-seq这一通用方法，可从FFPE样本中获取高质量的单细胞核转录组数据。我们将snPATHO-seq流程与现有针对冰冻或新鲜样本开发的10x 3’端及Flex检测方案进行性能基准对比，证实了三种流程产出的单细胞核RNA测序（single-nucleus RNA-seq, snRNA-seq）数据具有高度一致性。snPATHO-seq流程在覆盖多种正常与病变FFPE组织样本的测试中，同样展现出优异的鲁棒性。当与FFPE空间转录组技术（如FFPE Visium）结合使用时，snPATHO-seq可为FFPE样本提供多模态采样方案，助力实现更全面的转录组特征解析。

方法
冰冻外周血单个核细胞（peripheral blood mononuclear cell, PBMC）样本经解冻后，直接采用10x 3’端及Flex化学试剂进行建库处理。
针对冰冻乳腺癌组织样本，需先解离为单细胞核悬液，再采用10x 3’端及Flex化学试剂开展基因表达分析。
针对FFPE组织样本，分别采用snPATHO-seq流程或10x scFFPE流程提取细胞核，随后通过10x Flex化学试剂完成基因表达检测。
FFPE Visium数据按照厂商推荐流程，采用10x FFPE Visium CytAssist工作流进行构建。