ST-seq of homo sapiens: adult male lung cancer
RNAse is used to degrade mRNA in tissues, releasing probes that hybridize with mRNA. The polyA tail of the probe binds to poly(dT) sequences captured on the chip, and PCR amplification using the read2 sequence extends the probe, appending barcode and UMI sequences. Subsequently, using 0.08M KOH in an alkaline environment breaks hydrogen bonds between the extended probes and capture sequences, allowing probes with tagged sequences to transfer from solid-phase carriers to a liquid-phase system for convenient library construction. Single-stranded DNA is then PCR amplified to increase abundance and appended with sequencing adapters to complete library construction