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:
  1. Variable expression among different brain regions and cell types.
  2. Moderate abundance (too low: difficult to detect; too high: rolonies too crowded and difficult to resolve optically).
  3. Avoid alternatively spliced exons.
  • Considerations:
  1. Targeting RNA versus cDNA;
  2. Targeting nuclear transcripts versus cytoplasmic transcripts;
  3. Targeting 3'-ends or random positions;
  4. Gap versus no gap;
  5. Targeting single exons or across splice junctions;
  • Constrains:
  1. We can get only up to 240bp oligos from Agilent for now.
  2. A minimal list of features on the probes:
    1. Amplification adaptors (36-44bp, depending on the probe prep protocol).
    2. 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)
    3. Decoding region (6x20bp=120bp)
    4. Capturing arms (40-50bp, need to set an upper limit)

Rolonies generation

  • Considerations:
  1. Direct padlock capture versus whole transcriptome amplification

DNA in situ sequencing