Single Cell Fixed RNA Sequencing Service
Formalin-fixed paraffin-embedded (FFPE) is the primary method of sample preservation for clinical examinations and follow-ups, constituting a large and immensely valuable database of clinical information.
Over the past few years, high-throughput single-cell (nucleus) transcriptomics sequencing (scRNA/snRNA-seq) methods have revolutionized the entire field of biomedical research. Accurately identifying the transcriptomic features of each cell in clinical FFPE samples can lead to a better understanding of cellular heterogeneity and population dynamics, further improving the precision in diagnosing, treating, and prognosing human diseases. However, due to RNA cross-linking, modifications, and degradation during the FFPE preparation process, the isolation of single cells (nucleus) and RNA capture from FFPE tissues remain a challenge.
Technical Principles and Procedures
The main workflow of single-cell fixed RNA sequencing is shown in Figure 1. First, the obtained FFPE tissue undergoes dewaxing and rehydration, cell nucleus isolation, and permeabilization. To avoid genomic contamination, blocking primers are used to bind with the exposed single-strand DNA, and in-situ blocking is performed through multiple annealing and extension steps. Then, random primers and oligo(dT) primers enter the cell nucleus to bind to RNA, and in-situ reverse transcription of RNA molecules across the entire transcriptome is conducted. Poly(dA) is added in-situ to the 3' end of the cDNA by Terminal Deoxynucleotidyl Transferase (TdT). Subsequently, high-throughput single-cell nuclear labeling is carried out via a single-cell platform. Finally, lysis purification, cDNA amplification, second-generation library construction, and sequencing analysis are performed.
Fig.1 Technical principles and procedures of single-cell fixed RNA sequencing. (Xu, 2023)
Basic Performance
SnFixed-seq demonstrates extremely high cell marker specificity, with a twin cell rate of only 0.3% when detecting over 2000 cells. Similarly, UMI exhibits high species specificity (99%), indicating that snFixed-seq generates high-quality single-nucleus libraries. Additionally, a large number of long non-coding RNAs (lncRNAs) and short non-coding RNAs (sncRNAs), including small nucleolar RNAs (snoRNAs), small nuclear RNAs (snRNAs), and microRNAs (miRNAs) were detected (Figure 2c), suggesting that snFixed-seq can capture the entire transcriptome. The median single-nucleus gene count in 293T cell nuclei is 4141, with a UMI median of 11596, and an average sequencing depth of ~29k reads/cell; for 3T3 cell nuclei, the median single-nucleus gene count is 3427, the UMI median is 9795, and the average sequencing depth is ~25k reads/cell.
Fig.2 Performance testing of snRandom-seq was conducted using a human-mouse mixed cell line. (Xu, 2023)
Technique Specifications and Sample Requirements
- Cell Throughput Range: 1000-20000 cells/channel (standard reagent kit);
- Sample Throughput: 4 channels;
- Cell Size: No restrictions, both cells and nuclei are applicable;
- Cell Capture Rate: 50%~60%;
- Doublet Rate: Less than 2% per 5000 cells;
- Machine Time: No more than 10 minutes;
- Applicable Sample Types: Fresh, frozen, and FFPE samples;
- Applicable Species: Eukaryotes (humans, mice, rats, plants, etc.), and prokaryotes (bacteria, etc.).
Creative Biolabs' Single Cell Fixed RNA Sequencing Service
Creative Biolabs provides single cell Fixed RNA sequencing services, a cutting-edge technology offering a detailed perspective of the cellular landscape at the transcriptomic level. This technology excels in capturing comprehensive, full-length transcriptome data from FFPE samples with remarkable sensitivity through the use of random primers, significantly enhancing the detection of single-cell nuclear transcriptomes in FFPE and other degraded biological samples. When compared to conventional single-cell technologies, our single-cell fixed RNA sequencing stands out with its lower doublet rate, an increased count of identifiable genes, and consistent 5'-3' data coverage. Furthermore, its coverage surpasses that of two alternative FFPE snRNA-seq methods at both the single-cell and single-gene levels.
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Reference
- Xu, Z.; et al. High-throughput single nucleus total RNA sequencing of formalin-fixed paraffin-embedded tissues by snRandom-seq. Nature Communications. 2023, 14(1): 2734.
