![]() ![]() In this study, we develop snRandom-seq, a droplet-based high-sensitive and full-length snRNA sequencing method for FFPE tissues. ![]() Therefore, the overarching goal is to have an approach that can meet the need for high-throughput, high-sensitivity, and high-coverage snRNA-seq on FFPE tissues. In practice, large-scale and comprehensive transcriptomic profiling of clinical specimens is always required to identify predictive biomarkers or rare cell types. ![]() snFFPE-seq utilizes the poly(A)-based 10X Genomics platform, which is not sensitive enough to capture the low-quality RNAs from FFPE tissues 15. snPATHO-Seq depends on the probe-based 10X Genomics technology so that only limited genes can be detected 14. Two methods that were recently posted on bioRxiv, snPATHO-Seq 14, and snFFPE-seq 15, provided optimized methods to isolate single intact nuclei from FFPE tissues to perform snRNA-Seq, which demonstrates the feasibility of snRNA-Seq in FFPE tissues and unlocks a dimension of these hard-to-use samples. However, these methods were not yet workable for FFPE tissues. On the other hand, SPLiT-seq 11 was reported to be successfully used in fixed cells using random primer that was more efficient and broader to capture total RNAs 12, 13. SMART-seq-total 9 and VASA-seq 10 capture both polyadenylated and non-polyadenylated transcripts by deploying an extra step of tailing all RNA molecules with poly(A). Various methods have been developed to overcome these challenges from different perspectives. In addition, these oligo(dT)-based sc/snRNA-seq methods are primarily restricted to either fresh or fresh-frozen samples, as oligo(dT) primers usually fail on degraded RNAs. However, both single intact cell/nuclei isolation and RNA capture from FFPE tissues are still challenging due to RNA crosslinking, modification, and degradation.Ĭurrently, most popular high-throughput sc/snRNA-seq platforms, such as 10X Genomics Chromium Single Cell 3’ Solution, rely on oligo(dT) to capture poly(A) + RNAs, such that mainly matured messenger RNA (mRNA) will be detected rather than non-poly-adenylated RNAs for analysis. Accurate transcriptomics characterization of every single cell in clinical FFPE specimens is believed to have the ability to deliver a better understanding of cell heterogeneity and population dynamics, thereby improving the precision diagnostics, treatment, and prognosis of human disease. We have constructed the first human and mouse cell atlas with our customized high-throughput scRNA-seq platforms 7, 8. In the last few years, high-throughput single-cell/nuclei RNA sequencing (scRNA/snRNA-seq) methods have revolutionized the entire field of biomedical research 4, 5, 6. Recent studies have made great progress in transcription profiling in FFPE samples by optimal RNA extraction methods 2 or spatial in situ profiling 3. Inevitably, the irreversible modifications caused by formalin fixation on macromolecules in FFPE samples always make it challenging for molecular biology applications. The tissue morphology and cellular details of FFPE tissues are well-preserved for histopathology by the formaldehyde crosslinking among DNA, RNA, and proteins. Routine formalin-fixed paraffin-embedded (FFPE) tissues are the most common archivable specimens, constituting a vast and valuable patient material bank for clinical history, follow-up data, etc 1. ![]()
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