Kun:LabNotes/inSituSeq: Difference between revisions
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*Strategy: Experimentally evaluate the capture efficiencies of 13,000 probes, and pick the best ones. | *Strategy: Experimentally evaluate the capture efficiencies of 13,000 probes, and pick the best ones. | ||
*Choice of genes: | *Choice of genes: | ||
#Variable expression among different brain regions and cell types. | #Variable expression among different brain regions and cell types (based on published data in PMID: 22996553). | ||
#Moderate abundance (too low: difficult to detect; too high: rolonies too crowded and difficult to resolve optically). | #Moderate abundance (too low: difficult to detect; too high: rolonies too crowded and difficult to resolve optically). | ||
#Avoid alternatively spliced exons. | #Avoid alternatively spliced exons. |
Revision as of 23:24, 23 October 2012
RNA in situ sequencing
Responsibilities
- Rui: in situ rolony generation
- Matt: design and testing of decoding padlock probes
- Ho Suk: instrumentation (fluidic devices), automation, image analysis
Method developments
Probe design
- Strategy: Experimentally evaluate the capture efficiencies of 13,000 probes, and pick the best ones.
- Choice of genes:
- Variable expression among different brain regions and cell types (based on published data in PMID: 22996553).
- Moderate abundance (too low: difficult to detect; too high: rolonies too crowded and difficult to resolve optically).
- Avoid alternatively spliced exons.
- Considerations:
- Targeting RNA versus cDNA (RNA: one fewer step, more specific, efficiency could depends on secondary structure; DNA: one more step, can be immobilized, secondary structure could be less a problem);
- Targeting nuclear transcripts versus cytoplasmic transcripts (space available for imaging, integration with single-cell toto-RNAseq data);
- Targeting 3'-ends or random positions (affects how cDNAs are generated, poly-T primed or N6/9 primed);
- Gap versus no gap (Specificity versus sensitivity);
- Targeting single exons or across splice junctions (mature transcripts versus nascent transcripts or gDNA);
- Constrains:
- We can get only up to 240bp oligos from Agilent for now.
- A minimal list of features on the probes:
- Amplification adaptors (36-44bp, depending on the probe prep protocol).
- Common linker (48bp for our standard linker sequence, need to be reduced significantly, which is fine since we will be doing RCA instead of PCR and only a short annealing region is enough)
- Decoding region (6x20bp=120bp)
- Capturing arms (40-50bp, need to set an upper limit)
Rolonies generation
- Considerations:
- Direct padlock capture versus whole transcriptome amplification
DNA in situ sequencing
Can test the probes on RNA/cDNA first to estimate the efficiency.