Kun:LabNotes/inSituSeq: Difference between revisions
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===Method developments=== | ===Method developments=== | ||
====Probe design==== | ====Probe design==== | ||
*Strategy: Experimentally evaluate the capture efficiencies of 55,000 probes, and pick the best ones. | |||
*Choice of genes: | |||
#Variable expression among different brain regions and cell types. | |||
#Moderate abundance (too low: difficult to detect; too high: rolonies too crowded and difficult to resolve optically). | |||
#Avoid alternatively spliced exons. | |||
*Considerations: | *Considerations: | ||
#Targeting RNA versus cDNA; | #Targeting RNA versus cDNA; |
Revision as of 23:40, 9 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 55,000 probes, and pick the best ones.
- Choice of genes:
- Variable expression among different brain regions and cell types.
- 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;
- Targeting nuclear transcripts versus cytoplasmic transcripts;
- Targeting 3'-ends or random positions;
- Gap versus no gap;
- Targeting single exons or across splice junctions;
- 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