Figure 1. Overview of library preparation steps.

The library preparation can be divided in three parts in which the second part is performed by the robotic workstation. In part one, fresh frozen tissue sections are mounted on a barcoded array, and cDNA is synthesized from the mRNA in the tissue section. In part two, cDNA is transferred from the surface of the chip to the robot. The robot performs second strand synthesis and end repair, followed by in vitro transcription, adapter ligation and cDNA synthesis. Each step is accompanied by a reaction clean-up using paramagnetic carboxylic acid beads. Part three consists of sample indexing by PCR and, following clean-up, the sample is ready for sequencing.