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. 2023 Feb 23;41(10):1405–1409. doi: 10.1038/s41587-023-01676-0

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© The Author(s) 2023

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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Extended Data Fig. 1 — (a) ADT structure. The oligo labelled to the antibody has three functional regions: PCR handle (21 bp), antibody barcode (15 bp) and poly-A region (32 bp). (b) ADTs and mRNA with Poly-A region at the 3′ end can be reverse transcribed into cDNA using Barcode A as the RT primer. Barcode A consists of three functional regions, the poly-T region, spatial barcode region and the ligation region. During the first flow, 50 Barcode As were loaded into 50 parallel channels and the RT reaction was carried out inside each isolated channel (Step 1&2). After peeling off the 1st PDMS, a 2nd PDMS was attached. The in-channel ligation was performed with injecting 50 Barcode Bs into each of the 50 channels which are perpendicular to the channels of 1st PDMS chip (Step 3). Barcode B has four functional regions: ligation region, barcode region, UMI region and PCR handle region. Barcode B was also 5′ biotin modification. After ligation, tissue was lysed, and cDNAs were purified with streptavidin beads. The cDNAs on the beads were templated switched with template switch oligo (Step 4). PCR was used to amplify the cDNA (Step 5). The products were split into two portions, the mRNA derived cDNAs and the ADT derived cDNAs. The library was then built separately. More details were in the method section.