An automated approach to prepare tissue-derived spatially barcoded RNA-sequencing libraries

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. In this study, a protocol for retaining spatial information of transcripts has been adapted 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.


Installing and running the scripts Magnatrix 8000+ users
The protocol group file, including all scripts necessary to run the protocols has been packaged as a .Hxp file. It can be imported using the import function of the Magnatrix operating system. To import the protocol file, log in to the instrument with an Administrator or Power user account and start the Import Guide from the Tools menu. Follow the prompts on the screen and import the protocols to an AMF file of your choice. Restart the Magnatrix operating system and the new set of protocols should with all the scripts should appear in the chosen AMF file.
The imported Protocol Group consists of two programs to run the protocol in either 1-8 sample mode or 9-16 sample mode, each divided in two parts. The protocols intended to be used with the default settings, but it is possible to tweak settings such as incubation temperatures and incubation times. The protocol uses two Abgene 0600 PCR plates (ThermoFischer Scientific) and one Abgene 2.2 mL Storage plate.
Some small adjustments might be necessary to tailor the script to suit the conditions at other laboratories. The plate with enzymatic reagents are kept cool on one of the Peltier elements and, depending on the ambient temperature and humidity, there is a risk of condensation. It might be necessary to change the cooling temperature to prevent this (stored in the variable EnzymeTemp in the Variable files for each program). The script uses mainly relative movements, which should not require any modifications. However, when brushing the tips against the side of the liquid waste container the movement command is given in absolute coordinates, which might require adjustments. The coordinate is stored in the variable Some general points to take into consideration when re-writing the script for other platforms.
The platform must be able to perform magnetic separation of beads for the purification steps.
As the protocol consists of several enzymatic steps during the run it, is preferable to use a robotic workstation capable of keeping the reagents cool in one position while having another temperature-controlled unit for the enzymatic reactions. Reactions that are incubations at elevated temperatures are covered with oil to prevent evaporation losses. A system that has the capacity to seal plates can obviously omit oil and seal the plates instead. The aspiration volumes in the script have been adjusted depending on the temperature of the aspirated liquid in order to ensure that the correct volume is drawn and will need to be adjusted for a new liquid handler.

Supplementary Tables
Supplementary  M a n u a l 1 M a n u a l 2 M a n u a l 3 A u to m a te d 1 A u to m a te d 2 A u to m a te d 3 Number of unique transcripts M a n u a l 1 M a n u a l 2 M a n u a l 3 A u to m a te d 1 A u to m a te d 2 A u to m a te d 3 a b