A protocol for single-cell transcriptomics from cryopreserved renal tissue and urine for the Accelerating Medicine Partnership (AMP) RA/SLE network
Deepak A. Rao,
Celine C Berthier,
Edward P Browne,
Thomas M. Eisenhaure,
David J. Lieb,
Dawn E. Smilek,
James A. Lederer,
Michael B. Brenner,
E. Steve Woodle,
Jennifer H. Anolik,
Posted 05 Mar 2018
bioRxiv DOI: 10.1101/275859
Posted 05 Mar 2018
OBJECTIVE: There is a critical need to define the cells that mediate tissue damage in lupus nephritis. Here we aimed to establish a protocol to preserve lupus nephritis kidney biopsies and urine cell samples obtained at multiple clinical sites for subsequent isolation and transcriptomic analysis of single cells. METHODS: Fresh and cryopreserved kidney tissue from tumor nephrectomies and lupus nephritis kidney biopsies were disaggregated by enzymatic digestion. Cell yields and cell composition were assessed by flow cytometry. Transcriptomes of leukocytes and epithelial cells were evaluated by low-input and single cell RNA-seq. RESULTS: Cryopreserved kidney tissue from tumor nephrectomies and lupus nephritis biopsies can be thawed and dissociated to yield intact, viable leukocytes and epithelial cells. Cryopreservation of intact kidney tissue provides higher epithelial cell yields compared to cryopreservation of single cell suspensions from dissociated kidneys. Cell yields and flow cytometric cell phenotypes are comparable between cryopreserved kidney samples and paired kidney samples shipped overnight on wet ice. High-quality single cell and low-input transcriptomic data were generated from leukocytes from both cryopreserved lupus nephritis kidney biopsies and urine, as well as from a subset of kidney epithelial cells. CONCLUSION: The AMP RA/SLE cryopreserved tissue analysis pipeline provides a method for centralized processing of lupus nephritis kidney biopsies and urine samples to generate robust transcriptomic analyses in multi-center studies.
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