Spatially resolved transcriptomic profiling of degraded and challenging fresh frozen samples

Spatially resolved transcriptomics has enabled precise genome-wide mRNA expression profiling within tissue sections. The performance of methods targeting the polyA tails of mRNA relies on the availability of specimens with high RNA quality. Moreover, the high cost of currently available spatial resolved transcriptomics assays requires a careful sample screening process to increase the chance of obtaining high-quality data. Indeed, the upfront analysis of RNA quality can show considerable variability due to sample handling, storage, and/or intrinsic factors. We present RNA-Rescue Spatial Transcriptomics (RRST), a workflow designed to improve mRNA recovery from fresh frozen specimens with moderate to low RNA quality. First, we provide a benchmark of RRST against the standard Visium spatial gene expression protocol on high RNA quality samples represented by mouse brain and prostate cancer samples. Then, we test the RRST protocol on tissue sections collected from five challenging tissue types, including human lung, colon, small intestine, pediatric brain tumor, and mouse bone/cartilage. In total, we analyze 52 tissue sections and demonstrate that RRST is a versatile, powerful, and reproducible protocol for fresh frozen specimens of different qualities and origins.

All data analysis was carried out using the open source programming language R. Custom code used to run the analyses is available in our GitHub repository: https://github.com/ludvigla/RRST. A detailed specification of R package versions used for analyses can also be found in this repository. The code used to generate the figures as well as instructions for running the code inside a docker container are available at https://github.com/ludvigla/RRST.
All data required to replicate the analyses, including spaceranger output files, H&E images and additional files are available at Mendeley Data (DOIs for supplementary data: 10.17632/4w6krnywhn and 10.17632/442mhsrpcm.1).
Sequence data from the mouse brain and bone/cartilage samples have been deposited at GEO under accession number GSE221571. Sequence data for the pediatric brain tumors, colon/intestine, lung and prostate samples are available through a Materials Transfer Agreement with Monica Nister (monica.nister@ki.se), Guy Boeckxstaens (guy.boeckxstaens@kuleuven.be), Christos Samakovlis (Christos.Samakovlis@su.se) and Niklas Schultz (niklas.schultz@scilifelab.se), respectively in line with GDPR regulations. The data are available under Data Use 807 Conditions (DUO) and are limited to non-for-profit use as well as health/medical/biomedical 808 purposes. Access is granted if the above is fulfilled and local institutional review board/ethical 809 review board approvals are provided.
Sex and gender are not considered and important for this study.
Not relevant for this study.
No recruitment process was done in order to perform presented study. In this methodology paper, we aimed to demonstrate and benchmark our protocol of different tissue types. Individual samples were randomly provided from different ongoing projects. Patients operated for colorectal cancer or inflammatory bowel disease were recruited from a surgical ward with informed consent at University Hospitals Leuven. Lung tissues were obtained from deceased transplant organ donors by the Cambridge Biorepository for Translational Medicine (CBTM) with informed consent from the donor families and approval from the National Research Ethics Services (NRES) Committee of East of England. Patients who underwent radical prostatectomy were randomly selected by pathologist, written informed consent was obtained from all participants. Patients contributing with pediatric brain cancer samples were approached when operated for pediatric brain cancer. There were no biases in the selection procedure for all samples.
Use of prostate cancer samples was approved by the Regional Ethical Review Board (REPN) Uppsala, Sweden before study initiation (Dnr 2011/066/2, Landstinget Västmanland, Sari Stenius). Lung samples were obtained from deceased donors by the Cambridge Biorepository for Translational Medicine (CBTM) with informed consent from the donor families and approval from the NRES Committee of East of England -Cambridge South (15/ EE/0152), the project has received funding from the European Union's Horizon 2020 research and innovation programme under a grant agreement (no. 874656, discovAIR). GI tract specimens were approved by the medical ethics committee of University Hospitals Leuven (approval no. S62935). Use of pediatric brain tumor samples was approved by the Regional Ethical Review Board (EPN), Stockholm, Sweden (DNR 2018/3-31, Monica Nister).

March 2021
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Life sciences study design
All studies must disclose on these points even when the disclosure is negative. No sample size calculation was preformed. Samples were selected based on RNA quality and previously failed experiments in order to demonstrate presented protocol. 52 tissue sections of different biological origins analyzed in this manuscript showing the versatility of the method, while some sample processed in replicates demonstrate the robustness. Given the high number of processed sections and variety of samples representing different tissue types we believe the sample size is sufficient to demonstrate the usability of presented method.
No data were excluded from the analysis.
In order to verify the reproducibility of presented laboratory approach majority of the samples presented in this study were processed in technical replicates, where consecutive sections from the same tissue block were used to perform the experiments. In some cases biological replicates were used (sample from the same tissue from 2 different donors). In case of a mouse bone tissue, only one section per individual was used to perform standard Visium experiments due to the difficulty to obtain good quality data and the price for each experiment. All details can be found in Table 1 and Supplementary Data 1.
Randomization of samples is not relevant for this study. In this work, samples were chosen based on RNA quality in order to perform comparison of 2 protocols, next samples were chosen based on biological origin to validate the method across different tissue types. As presented work is focused on method the randomization of samples/subjects is not of an importance in order to demonstrate its performance. Data analysis of all samples in this study was performed in groups according to the tissue origin (organ).
Blinding was not relevant for this study. Samples from the same tissue specimens were analyzed together in order to make fair comparisons as each tissue is composed from different cell types that presents different challenges in sample processing and data generation.
Mouse brain samples were from C57BL/6J male mice of age 2 months and were commercially purchased from Adlego Biomedical. Mouse bone and cartilage samples were collected from postnatal mice (C57/BL6) at four and eleven days of age, sex of mice was not recorded due to difficulties to sex determination in this early stages of development. Mice were group-housed with the parent mouse on a 12h light-dark cycle, at 22°C with 50% humidity in the air.