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:
- 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)
- First round probe design.
- A list of 5333 genes were generated by Dr. Yun Zhu from Wei Wang's group based on the following criteria:
- Genes that show variable expression in the brain (based on PMID: 22996553).
- Genes included in the ABI's ISH data.
- Genes present in both cytosol and nucleus (based on the ENCODE RNAseq paper).
- Matt selected up to 5 candidate regions per gene for probe design. One set of probes was designed with a gap size of 20bp. A second set has no gap.
- I wrote a script to filter the probes to keep:
- Up to 5 probes per gene.
- All genes with names like "LOCxxxx" or "CxxOrfxxxx" are excluded because they are poorly annotated.
- I then double-checked these probes by mapping to the human genome and reference mRNA sequences using NovoAlign.
- A list of 5333 genes were generated by Dr. Yun Zhu from Wei Wang's group based on the following criteria:
/home/kunzhang/softwares/Novocraft/novocraft/novoalign -d /home/kunzhang/RNAseq/Data/CommonFiles/refMrna.ndx -f outputFile_0gap_filtered.fasta -F FA -r ALL > outputFile_0gap_filtered_novoalign_refMrna.out /home/kunzhang/softwares/Novocraft/novocraft/novoalign -d /home/kunzhang/softwares/Novocraft/novocraft/human_g1k_v37 -f outputFile_0gap_filtered.fasta -F FA -r ALL > outputFile_0gap_filtered_novoalign_human_g1k_v37.out & /home/kunzhang/softwares/Novocraft/novocraft/novoalign -d /home/kunzhang/RNAseq/Data/CommonFiles/refMrna.ndx -f outputFile_20gap_filtered.fasta -F FA -r ALL > outputFile_20gap_filtered_novoalign_refMrna.out /home/kunzhang/softwares/Novocraft/novocraft/novoalign -d /home/kunzhang/softwares/Novocraft/novocraft/human_g1k_v37 -f outputFile_20gap_filtered.fasta -F FA -r ALL > outputFile_20gap_filtered_novoalign_human_g1k_v37.out &
- All probes that were not mapped to the refMrna, or were mapped to more than one location in the genome are excluded.
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.
- Since we will start with ~5000 candidate genes, more barcoded is needed. For this I increased the number of colors (fluorophores) to 3. With only 7 hybridization stages, we can decode 5040 genes. I also added another constrain that each barcode needs a minimal of two colors. The barcodes are also shorter, which gives more flexibility for the design of the common linker.
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.