Targeted ExSeq -- Sequencing Library Preparation v1

This protocol accompanies Expansion Sequencing (ExSeq), describing the process of targeted ExSeq library preparation for a sample that has been processed according to a Targeted ExSeq Tissue Preparation protocol. The steps described here are a generalization of the protocol used in figures 4-6 of the paper, and represent our recommendations for future users of the technology. The flowchart in Fig. 1A depicts the library preparation workflow. Fig. 1B is a summary of the product, in which padlock probes are amplified to form amplicon concatamers. The net result of the process is that barcode sequences are delivered to transcripts of interest and locally amplified hundreds to thousands of times. The process of library preparation encompasses the following steps. (1) Oligonucleotide padlock probes bearing barcode sequences hybridize to RNA transcripts of interest (Step 8). (2) SplintR Ligase, which can has RNA-splinted DNA ligase activity, ligates adjacent ends of padlock probes, forming circular DNA molecules (Step 9). (3) A universal primer hybridizes to all padlock probes (Step 10). (4) Rolling Circle Amplification initiates from the primers, and repeatedly copies the sequence of the padlock probes, forming an amplicon (Step 11). (5) BS(PEG)9 covalently cross-links the amplicon to itself, stabilizing the amplicon (Steps 12-13) during in situ sequencing. At this point, the sample is now ready for downstream detection (i.e. via hybridizing fluorophore-labeled oligos to the amplicon (Step 14), and in situ sequencing). This protocol was used (with no modifications) to profile human metastatic breast cancer biopsies as a part of the Human Tumor Atlas Pilot Project (HTAPP).