Sequencing the nucleic acid content of individual cells or specific biological samples is becoming increasingly common. This drives the need for robust, scalable and automated library preparation protocols. Furthermore, an increased understanding of tissue heterogeneity has lead to the development of several unique sequencing protocols that aim to retain or infer spatial context.
Researchers from the Royal Institute of Technology, Sweden adapted a protocol for retaining spatial information of transcripts to run on a robotic workstation. The method spatial transcriptomics is evaluated in terms of robustness and variability through the preparation of reference RNA, as well as through preparation and sequencing of six replicate sections of a gingival tissue biopsy from a patient with periodontitis. The results are reduced technical variability between replicates and a higher throughput, processing four times more samples with less than a third of the hands on time, compared to the standard protocol.
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.