Kun:LabNotes/inSituSeq: Difference between revisions
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(Created page with "==RNA in situ sequencing== ===Responsibilities=== *Rui: in situ rolony generation *Matt: design and testing of decoding padlock probes *Ho Suk: instrumentation (fluidic device...") |
m (→Probe design) |
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#Gap versus no gap; | #Gap versus no gap; | ||
#Targeting single exons or across splice junctions; | #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==== | ====Rolonies generation==== | ||
*Considerations: | *Considerations: | ||
#Direct padlock capture versus whole transcriptome amplification | #Direct padlock capture versus whole transcriptome amplification | ||
==DNA in situ sequencing== | ==DNA in situ sequencing== |
Revision as of 23:36, 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
- 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