SUMMARY
Done in wet lab.
- We extract DNA from cells.
- Purification column
- Centrifuge
- How does this work?.. high-level overview
- DNA and RNA are split, and RNA is reverse-transcribed into DNA
- DNA fragmentation is carried out via sonication (very small, fast vibration breaking apart cells enzymatically with catalysts.
- Adapter sequences are added to both ends of DNA fragments. They allow the DNA fragments to bind to the flow cell (the specialized surface where sequencing reactions occur).
- The library (a collection of short DNA fragments with adapters) is loaded onto a flow cell. Each fragment binds to the flow cell and then undergoes bridge amplification, creating many copies of the same fragment to ensure strong signal.
- All copies from same cell carry same UMI (Unique Molecular Identifier, a short random DNA). We can group those to remove amplification effect when calculating real expression.
- Sequencing by synthesis is when we read the DNA. Sequencing primers bind to the adapter sequences on the DNA clusters.
- In each sequencing cycle, a mix of modified nucleotides (A, T, C, G) is added. These nucleotides have
- fluorescent tag: each type of base (A, T, C, G) is labeled with its own colored fluorophore (a molecule that absorbs light energy and re-emits it as a fluorescence)
- terminator: ensure only one nucleotide can be added to the DNA strand at a time
- After addition of one abuse, the sequencer takes an image. The cameras detect the specific fluorescent color emitted from each cluster, identifying the added base.
- Then, a chemical cleavage step removes the fluorescent dye and the reversible terminator, allowing the next nucleotide to be added in the subsequent cycle.
- This cycle of adding a single base, imaging, and cleaving (breaking a particular chemical bond) is repeated millions of times (e.g. 100-300 cycles), building up the whole DNA sequence.
The images from each cycle are processed to generate raw sequence reads (FASTQ files), which are then aligned to a reference genome.
High quality RNA are needed for mRNA libraries. Degraded samples should only be used to make a “total” RNA-seq library (rRNA removal).
RNA enrichment
- polyA tailed messenger RNA: mRNA-seq
- total RNA (rRNA removed): “total” RNA-seq
mRNA (polyA) purification is done via mRNA enrichment: nRMA binds beads coated with oligo dT primer and non-polyadenylated transcripts are washed away. Transcripts lost in polyA purification:
- Ribosomal/Transfer RNA
- Histone mRNA
- Long-noncoding RNA
- Nascent intron containing transcripts
- Micro RNA
- Degraded RNA
- Many viral transcripts
- Prokaryote/Bacterial transcripts
- polyA is the degradation signal
If polyA tail is no longer attached to transcript, it results in differential loss of transcripts between samples.
NOTE
PCR (Polymerase Chain Reaction) A lab technique to make millions of copies of a specific DNA fragment.