Interest in studying the spatial distribution of gene expression in tissues is rapidly increasing. Spatial Transcriptomics is a novel sequencing-based technology that generates high-throughput information on the distribution, heterogeneity and co-expression of cells in tissues. Unfortunately, manual preparation of high-quality sequencing libraries is time-consuming and subject to technical variability due to human error during manual pipetting, which results in sample swapping and the accidental introduction of batch effects. All these factors complicate the production and interpretation of biological datasets.
Researchers at the KTH Royal Institute of Technology, Sweden have integrated an Agilent Bravo Automated Liquid Handling Platform into the Spatial Transcriptomics workflow. Compared to the previously reported Magnatrix 8000+ automated protocol, this approach increases the number of samples processed per run, reduces sample preparation time by 35%, and minimizes batch effects between samples. The new approach is also shown to be highly accurate and almost completely free from technical variability between prepared samples.
Workflow for automated ST library preparation
a Each ST barcoded array contains six subarrays, each with 2000 100-μm spots. Every spot contains oligo-dT probes bearing a spot-specific barcode. The protocol is divided into four parts. The first is performed on the chip, where fresh frozen tissue sections are mounted on the barcoded subarrays. The tissue sections are permeabilized, allowing their mRNA to be captured by the oligo-dT probes on the surface, which function as primers for overnight cDNA synthesis. On the following day, the tissue sections are removed from the subarray surface. The cDNA-mRNA hybrids are then released and collected per sample and transferred to the Bravo system for the second and third parts of the protocol. Finally, the libraries undergo PCR indexing in parallel before sequencing. b Graphical interface to the automated program. c Layout of the Bravo working deck prior to start. Positions A to C are used for tips and waste, while the reaction plate, containing the input material together with master mixes for the enzymatic reactions is placed on position D, which is kept at 4 °C. On position E, a 2 mL deep well plate is holding the reagents for the bead clean up steps. An empty 96-well plate is placed on the temperature controlled position F, which is where the enzymatic reactions are carried out. Positions G to H are used during reaction clean up
The new automated Spatial Transcriptomics protocol using the Agilent Bravo Automated Liquid Handling Platform rapidly generates high-quality Spatial Transcriptomics libraries. Given the wide use of the Agilent Bravo Automated Liquid Handling Platform in research laboratories and facilities, this will allow many researchers to quickly create robust Spatial Transcriptomics libraries.