Figure 1

Molecular steps to generate sequencing substrate in targeted and non-targeted in situ sequencing. (a) In the targeted approach, a specific or random primer is used to retrotranscribe mRNA (gray) (i), the synthesized cDNA is made single stranded and a padlock probe is hybridized to complementary target sequences on the cDNA molecule (ii, iii), leaving a short gap between the probe arms (in red). Polymerization gap filling and DNA ligation are used to circularize the padlock probe (iv), which can be then replicated via target-primed rolling circle amplification (v). (b) In non-targeted in situ sequencing, the cDNA is synthesized by random primers containing a sequencing adaptor (vi). RNA is then digested and single-stranded cDNA molecules are self-circularized (ii, iii). An external primer is then hybridized and used to prime the rolling circle amplification (RCA) reaction (ix, x). In both the approaches, the sequencing substrate consists of a rolling circle product containing a clonally amplified region of DNA (colored in red or green), which is sequenced in situ by sequencing using ligation chemistries.