Fig. 2
Description of the experimental process for RNA sequencing in single cells. A suspension of cells (A) is used to separate each cell into individual reactors. The figure describes the approach based on the formation of microbubbles (B), which contain beads to allow the capture of RNA released by cell lysis within the reactor (C). RNA capture by the bead in the reactor is permitted by covering the beads with oligonucleotides that have a T-tail in their 3’ terminal portion (D). Each bead contains millions of these oligonucleotides that have common sequences (oligod(T) and R1), a unique sequence for each oligonucleotide (UMI) and a different sequence for each bead (cell barcode) (E). The UMIs allow for the digital counting of gene expression because different UMIs can be associated with the same RNA sequence, based on the abundance of the RNA. The cell barcode allows understanding whether the RNAs being evaluated are derived from the same bead, and therefore cell, or from different beads, and therefore different cells. Oligod(T) allows the capture of polyadenylated RNAs and their retrotranscription (F). Once retrotranscriptase (RT) reaches the 3’ terminal of RNA, due to its 3’ terminal transferase activity, it adds 3 cytosines to the cDNA (G). The overhang of C allows binding of the template switch primer (TSP) and continuation of retrotranscription of this primer (H) as well. At the end of retrotranscription, the cDNA will be characterized by having known ends given by R1 and the template switch primer (I and J). This whole process takes place within each reactor and therefore separately for each cell. Since at the end of the retrotranscription the cDNAs will be labeled with the cell barcode, it is possible to destroy the bubbles and continue the rest of the protocol in bulk. To amplify the low amount of RNA released from each individual cell, a PCR is done by exploiting primers complementary to R1 and TSP (K). This will yield a dsDNA with two known ends (L) that can be used for the sequencing of the full length by binding sequencing adapters to the ends of cDNA by using a PCR or can be fragmented and sequenced the portion corresponding to the 3’ end of RNA.