What is full-length sequencing?
With the development of long-read RNA sequencing, it is now possible to sequence cDNA transcripts in their entirety—without assembly. These full-length sequences improve genome annotation and provide a way to look at gene expression data in an isoform- or allele-specific way.
How do you get full-length cDNA?
The full-length cDNA can be directly amplified from the pool of adaptor-ligated cDNA by LA PCR using two gene- specific primers from the extreme 5′ and 3′ ends of the cDNA sequence (5′ GSP and 3′ GSP, Figure 1).
How do you sequence cDNA?
- Finding cDNA sequence for a gene. Step 1 – Search. Step 2 – Choose a transcript. Step 3 – Access the cDNA sequence.
- Using a sequence to find a gene (BLAST/BLAT) Step 1 – Using BLAST/BLAT. Step 2 – View the results. Step 3 – Viewing the hit.
What is the length of the cDNA?
The average cDNA insert size was 1.9 kb. Approximately, 96% of the clones had inserts longer than 500 bp, 54% of the cDNA clones had inserts longer than 1.5 kb, and 15% of the clones contained inserts longer than 3 kb.
What do I need for full length cDNA sequencing?
For full length cDNA sequencing we require 1 ug of total RNA in up to 20 ul of molecular biology grade water. The RNA samples need to be DNA-free and need to be accompanied by Bioanalyzer traces. The samples should have RNA-integrity scores (RIN-scores) of 8 or higher.
Where is the artificial sequence located on a cDNA?
Every full-length cDNA carries the entire 5′ end of the transcript and an additional artificial sequence, which in this case is the same as the one located at the 5′ end of the oligo-dT primer (i.e., the 3′-end of the mRNA), making possible the subsequent PCR using a single primer.
How is smart Seq2 used for cDNA generation?
Here we present a detailed protocol for Smart-seq2 that allows the generation of full-length cDNA and sequencing libraries by using standard reagents. The entire protocol takes ∼ 2 d from cell picking to having a final library ready for sequencing; sequencing will require an additional 1–3 d depending on the strategy and sequencer.
How is rapid amplification of cDNA ends used?
Rapid amplification of cDNA ends. Rapid amplification of cDNA ends (RACE) is a technique used in molecular biology to obtain the full length sequence of an RNA transcript found within a cell.