Kun:LabNotes/inSituSeq: Difference between revisions
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##Capturing arms (40-50bp, need to set an upper limit) | ##Capturing arms (40-50bp, need to set an upper limit) | ||
====Linker design==== | ====Linker design==== | ||
*I wrote a [[Media:get_decoding_linkers.txt|script]] to generate a list of barcoded linker sequences. A total of 1792 linkers were generated using two colors and eight stages. | *I wrote a [[Media:get_decoding_linkers.txt|script]] to generate a list of barcoded linker sequences. A total of [[Media:decoding_linkers_8-stages_2-colors.txt|1792 linkers]] were generated using two colors and eight stages. | ||
====Rolonies generation==== | ====Rolonies generation==== |
Revision as of 18:55, 30 November 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 6,500 probes(or 13,000, depending on the minimal order allowed by Agilent), and pick the best ones.
- Choice of genes:
- Variable expression among different brain regions and cell types (based on published ABI Brain Atlas data).
- Moderate abundance (too low: difficult to detect; too high: rolonies too crowded and difficult to resolve optically).
- Avoid alternatively spliced exons.
- Prioritizing genes used in ABI ISH mapping: Disease genes (383); Human cortical marker genes (167, 55 overlapped with disease genes).
- 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), nucleus versus cytoplasmic transcripts (based on the ENCODE RNAseq paper).
- 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)
Linker design
- I wrote a script to generate a list of barcoded linker sequences. A total of 1792 linkers were generated using two colors and eight stages.
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.