Kun:LabNotes/inSituSeq

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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:
  1. Variable expression among different brain regions and cell types (based on published ABI Brain Atlas data).
  2. Moderate abundance (too low: difficult to detect; too high: rolonies too crowded and difficult to resolve optically).
  3. Avoid alternatively spliced exons.
  4. Prioritizing genes used in ABI ISH mapping: Disease genes (383); Human cortical marker genes (167, 55 overlapped with disease genes).
  • Considerations:
  1. 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);
  2. 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).
  3. Targeting 3'-ends or random positions (affects how cDNAs are generated, poly-T primed or N6/9 primed);
  4. Gap versus no gap (Specificity versus sensitivity);
  5. Targeting single exons or across splice junctions (mature transcripts versus nascent transcripts or gDNA);
  • 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

Can test the probes on RNA/cDNA first to estimate the efficiency.