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. 2017 Oct 11;7:12941. doi: 10.1038/s41598-017-13462-5

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Schematic of spatially resolved gene expression analysis on adult heart. (a) In order to establish optimal conditions for retrieval and attachment of mRNA in situ, a quality control assay is first used. Intensity of signals generated from the Cy3-cDNA footprint is then scored to examine optimal permeabilization conditions. (b) A spatially barcoded microarray, printed with 1,007 clusters (i.e features) of reverse-transcription oligo-dT primers, are then used with established permebilization conditions. Each feature on the array contains more than 200 million probes, all sharing a unique DNA sequence (barcode) specific to that feature. The barcode is used in the downstream analysis to link each feature’s position within the tissue to the mRNA captured at that position. Finally, following reverse transcription and tissue removal, barcoded cDNA is enzymatically released from the array and used to generate sequencing libraries.