Fig. 6

Technologies for discover of RNA location. a Schematics of FISSEQ and STARmap. FISSEQ begins with fixing cells on a glass slide and performing reverse transcription in situ with aminoallyl-dUTP and adapter sequence-tagged random hexamers. After RT, cDNA fragments are circularized at 60 °C. The circular templates are amplified using rolling-circle amplification (RCA) primers complementary to the adapter sequence in the presence of aminoallyl-dUTP and stably cross-linked. The nucleic acid amplicons in cells are then ready for sequencing and imaging. STARmap begins with labeling of cellular RNAs by pairs of DNA probes followed by enzymatic amplification so as to produce a DNA nanoball (amplicon). Tissue can then be transformed into a 3D hydrogel DNA chip by anchoring DNA amplicons via an in situ—synthesized polymer network. b Schematics of biochemical cell fractionation. Biological extracts including intact organelles are separated by density gradient or immunoprecipitation with specific antibodies. c Overview of the APEX-RIP. Cells expressing APEX2 targeted to the mitochondrial are cultured with the APEX substrate biotin-phenol. H₂O₂ initiates biotinylation of proximal endogenous proteins, which are subsequently crosslinked to nearby RNAs by formaldehyde. After cell lysis, biotinylated species are enriched by streptavidin pull-down, and coeluting RNAs are analyzed by RNA-Seq