Scalable in situ single-cell profiling by electrophoretic capture of mRNA using EEL FISH
). Each probe consisted of a 26- to 32-nucleotide-long sequence, reverse complementary of the target RNA sequence, and one or two overhanging tails containing the six detection sequences of 20 nucleotides each, to which the detection probes can bind. These six sequences were added to the RNA-binding sequence in a random order to reduce the potential steric hindrance effect on signal intensity when they accumulate over sequential rounds of hybridization.
Encoding probes were ordered either from IDT as oPools at ready-to-use concentrations or as Twist Bioscience Oligo Pools for the 448-gene mouse and human experiments. For higher-complexity probe sets, Twist Bioscience Oligo Pools were used and amplified using a previously published protocolwith the following adaptations. We performed an initial PCR amplification to generate an intermediate stock and used it as the template for the subsequent experiment, not to exhaust the original probe pool. Then, the probes were amplified again by PCR, followed by in vitro transcription, after which the RNA was purified using the Zymo Oligo Clean and Concentrator.
Hoechst for the mouse atlas experiments, followed by five washes with 2× SSC. The sample was covered with a 24 × 32 mm coverslip spaced by a parafilm gasket, and an overview image of the sample was taken using a ×10 objective. The area of the sample was traced on the overview image and used to generate field of view positions for the ×40 and ×60 objectives to cover the entire sample.
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