LungMAP2 URMC cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) protocol v1

The LungMAP program has been working for over a decade to provide the lung community with a comprehensive resource for the lung research community, including reference data sets and protocols [1]. The University of Rochester has served as the Human Tissue Core and worked extensively with Research Center lab groups. We have established protocols for procuring human tissue including, but not limited to lung, and processing them for a variety of downstream analysis modalities including the generation of single cell suspensions [2, 3, 4]. We have successfully utilized singe cells suspensions to generate single cell CITESeq data utilizing the 10X Genomics Single Cell 3' (v3.1) RNA sequencing platform [5-7]. This protocol describes the steps necessary for preparing generating sequencing data following staining single cells with ADT-tagged antibodies (including library preparation, sequencing steps, and mapping steps). References 1. https://www.lungmap.net/about-lungmap/lungmap-objectives/ 2. 602.2 Donor Acceptance Criteria for URMC HTC SenNet Inclusion dx.doi.org/10.17504/protocols.io.biz7kf9n. 3. 603.3 & 604.5_URMC_HTC_Lung and Lobe Processing for SenNet dx.doi.org/10.17504/protocols.io.n2bvj395plk5/v1 4. 702.B.3 URMC HTC Lung Tissue Digestion SOP + Worksheet 052820 dx.doi.org/10.17504/protocols.io.biz5kf86 5. 709.1 Staining of Dissociated Lung Cells for Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE sequencing) V.2 dx.doi.org/10.17504/protocols.io.e6nvw1jk7lmk/v2 6. TotalSeq‱-A Antibodies and Cell Hashing with 10x Single Cell3' Reagent Kit v3 3.1 Protocol dx.doi.org/10.17504/protocols.io.8aahsae 7. Chromium Next GEM Single Cell 3’ Reagent Kits v3.1(Dual Index) User Guide. Document Number: CG000315. October 2022.