To implement the two-layer DNA seqFISH+ strategy, 96 unique readout probes were used in each fluorescent channel for a total of 288 unique readout probes for 3 fluorescent channels. The readouts probe sequences were obtained from our previous DNA seqFISH+ studies7 (link),8 (link) as well as additional orthogonal readout probe sequences were generated and validated similarly to those previous studies. The RNA seqFISH+ readout probe sequences were selected from a subset of the two-layer DNA seqFISH+ readout probe sequences. The readout probe sequences (12-15-nt) for sequential immunofluorescence were selected from our previous studies7 (link),8 (link) and further designed and validated with the same criteria for this study. The fluorescently-labeled readout probes (Integrated DNA Technologies) that can bind to the readout sequences on the primary probes or primary antibodies were conjugated in-house to Alexa Fluor 647–NHS ester (Invitrogen A20006), Cy3B–NHS ester (GE Healthcare PA63101), or Alexa Fluor 488–NHS ester (Invitrogen A20000) as described before17 (link) or directly purchased (Integrated DNA Technologies).
Protocol detail
Multiplexed Spatial Transcriptomics via DNA seqFISH+
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Alexa fluor 488
Alexa fluor 647
Antibodies
Cy3b nhs ester
Ester
Fluorescent probes
Immunofluorescence
Probe dna
Corresponding Organization : California Institute of Technology
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Variable analysis
independent variables
- Use of 96 unique readout probes in each fluorescent channel for a total of 288 unique readout probes for 3 fluorescent channels
- Not explicitly mentioned
- Readout probe sequences (12-15-nt) for sequential immunofluorescence selected from previous studies and further designed and validated with the same criteria for this study
- Fluorescently-labeled readout probes conjugated in-house to Alexa Fluor 647–NHS ester, Cy3B–NHS ester, or Alexa Fluor 488–NHS ester as described before or directly purchased
- Positive controls: Not explicitly mentioned
- Negative controls: Not explicitly mentioned
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