Beskrivelse
The HieffTM LongSeq cDNA Library Prep Kit utilizes Oligo (dT) Primers to initiate reverse transcription. Leveraging the terminal transferase activity (Template-switching activity) of the reverse transcriptase, a specific sequence is added to the 3' end of the cDNA. Subsequent PCR amplification employs primers targeting this specific sequence and the terminal portion of the Oligo (dT) Primer to generate full-length cDNA.
Components
|
Components No. |
Name |
12937ES08 |
12937ES24 |
12937ES96 |
|
|
12937-A |
|
Oligo (dT) Primer |
8 μL |
24 μL |
96 μL |
|
12937-B |
|
5×RT Buffer |
32 μL |
96 μL |
384 μL |
|
12937-C |
|
1st Strand Enzyme Master Mix |
16 μL |
48 μL |
192 μL |
|
12937-D |
|
TSO Primer |
8 μL |
24 μL |
96 μL |
|
12937-E |
|
2nd Reaction Buffer |
56 μL |
168 μL |
672 μL |
|
12937-F |
|
2nd Strand Enzyme Mix |
24 μL |
72 μL |
288 μL |
|
12937-G |
|
2×Multiplex Long PCR Master Mix |
200 μL |
600 μL |
2×1200 μL |
|
12937-H |
|
PCR-F |
20 μL |
60 μL |
240 μL |
|
12937-I |
|
PCR-R |
20 μL |
60 μL |
240 μL |
Storage
This product should be stored at -25~-15℃ for 1 year.
Applications of Full-length Transcriptome Sequencing
1. Agriculture
- Refining Transcriptome Information: Analysis of complex gene structures, as well as transcript structure and function.
- Differential/Dynamic Transcriptomics: Investigating transcriptional changes under stress treatments or across different time sequences; studying the mechanisms of animal and plant domestication.
- Comparative Transcriptomics: Analyzing phylogenetic relationships and mRNA sequence differences between closely related species.
2. Medicine
- Fusion Gene Identification: Elucidating the pathogenic mechanisms of fusion genes in cancer.
- Complex Gene Transcriptome Research: Studying complex splicing forms of disease- or cancer-related genes.
- Quantitative Transcriptomics: Quantification at the gene/transcript level to mine for condition-specific genes and transcripts.
Application Cases
1. UHRR Standard Data Demonstration
Experimental Protocol: Libraries were constructed using the Yeasen Full-length Transcriptome Kit (12937ES) and sequenced on the CycloneSEQ G400-ER / G100-ER nanopore platform.
Data Performance: With a total throughput exceeding 248 Gb, the full-length transcript ratio reached 88.02%, and the valid mapping rate was 98.01%.
|
Total Output(Gb) |
Total Reads (M) |
Full-Length Transcript Output(Gb) |
Full-Length Output (%) |
Full-Length Reads (%) |
Mapping Rate (%) |
|
240.75 |
221.89 |
211.91 |
88.02 |
86.17 |
98.01 |
Key Results: The data demonstrates stable, high-quality recovery of full-length transcripts. As read counts increase, the detection of genes and transcripts improves continuously. Furthermore, coverage is uniform from the Transcription Start Site (TSS) to the Transcription End Site (TES), showing no significant 5' or 3' bias, which supports precise transcript structure analysis.
2. Real Sample Data Demonstration
Experimental Protocol: Utilizing the Yeasen Full-length Transcriptome Kit (12937ES) combined with the CycloneSEQ G400-ER / G100-ER platform.
Sample Diversity: The study included diverse samples such as mouse spleen, mouse liver, male/female zebrafish, UHRR, and yeast, with 4 biological replicates for each.
|
Sample |
Replicate |
Full-length(%) |
Valid Base(%) |
Mapping(%) |
N50(Kb) |
Sense Strand(%) |
Antisense Strand(%) |
|
UHRR |
1 |
84.66 |
84.18 |
98.10 |
1.96 |
50.82 |
49.18 |
|
UHRR |
2 |
84.65 |
84.14 |
98.15 |
1.94 |
51.57 |
48.43 |
|
UHRR |
3 |
81.98 |
81.16 |
98.08 |
1.88 |
51.41 |
48.59 |
|
UHRR |
4 |
81.72 |
80.90 |
98.00 |
1.87 |
51.24 |
48.76 |
|
Yeast |
1 |
87.10 |
88.37 |
96.69 |
1.55 |
51.73 |
48.27 |
|
Yeast |
2 |
86.52 |
87.72 |
96.77 |
1.54 |
52.03 |
47.97 |
|
Yeast |
3 |
86.01 |
87.14 |
96.74 |
1.54 |
51.31 |
48.69 |
|
Yeast |
4 |
87.05 |
88.39 |
96.73 |
1.54 |
51.86 |
48.14 |
|
Mouse Liver |
1 |
86.05 |
87.36 |
98.34 |
1.70 |
50.83 |
49.17 |
|
Mouse Liver |
2 |
86.08 |
87.28 |
98.21 |
1.71 |
50.96 |
49.04 |
|
Mouse Liver |
3 |
87.09 |
88.55 |
98.38 |
1.71 |
50.73 |
49.27 |
|
Mouse Liver |
4 |
86.38 |
87.28 |
98.28 |
1.71 |
50.50 |
49.50 |
|
Mouse Spleen |
1 |
84.83 |
84.51 |
98.11 |
1.80 |
50.82 |
49.18 |
|
Mouse Spleen |
2 |
84.12 |
83.33 |
98.97 |
1.83 |
50.80 |
49.20 |
|
Mouse Spleen |
3 |
83.70 |
82.91 |
97.97 |
1.85 |
50.91 |
49.09 |
|
Mouse Spleen |
4 |
84.36 |
84.30 |
98.20 |
1.80 |
50.82 |
49.18 |
|
Zebrafish (Female) |
1 |
86.74 |
88.34 |
96.57 |
1.49 |
50.89 |
49.11 |
|
Zebrafish (Female) |
2 |
85.75 |
86.68 |
96.93 |
1.46 |
51.15 |
48.85 |
|
Zebrafish (Female) |
3 |
87.01 |
88.04 |
96.75 |
1.46 |
51.12 |
48.88 |
|
Zebrafish (Female) |
4 |
87.18 |
88.59 |
96.95 |
1.47 |
50.81 |
49.19 |
|
Zebrafish (Male) |
1 |
84.99 |
85.60 |
97.19 |
1.64 |
51.03 |
48.97 |
|
Zebrafish (Male) |
2 |
85.00 |
85.54 |
97.33 |
1.63 |
51.04 |
48.96 |
|
Zebrafish (Male) |
3 |
83.75 |
83.82 |
97.36 |
1.62 |
51.57 |
48.43 |
|
Zebrafish (Male) |
4 |
84.40 |
84.55 |
97.13 |
1.63 |
51.62 |
48.38 |
Key Results: Across all species and varying complexities, the data showed high stability and reproducibility. The full-length transcript ratio exceeded 83% for all samples, with mapping rates consistently above 96.5%.
Documents:
Safety Data Sheet
Manuals
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