Single-nucleus RNA sequencing of human fetal lung from 4 anatomic regions
Single-nucleus RNA sequencing of human fetal lung from 4 anatomic regions
Human lung tissue research was reviewed and approved by The University of Michigan Institutional Review Board (IRB). Normal, de-identified human fetal lung tissue was obtained from the University of Washington Laboratory of Developmental Biology. Specific tissue regions were dissected according to anatomic region and snap-frozen in liquid nitrogen for nuclei isolation. Nuclei were isolated from the tissue using the standard protocol for the 10X Chromium Nuclei Isolation Kit(10X Cat#1000437) by the authors. Nuclei were submitted to the University of Michigan Advanced Genomics Core for 10X On-Chip Multiplexing (OCM). Four spatially distinct regions were microdissected from a single lung specimen and processed independently to generate single-cell suspensions. The resulting suspensions were subsequently multiplexed using 10x Genomics Chromium On-Chip Multiplexing (OCM). Following OCM labeling, cells from all specimens were pooled prior to GEM generation and library construction according to the manufacturerâ€™s protocol. Because multiplexing occurred before library generation, a single sequencing library and corresponding raw FASTQ files were produced for the pooled sample. 5000 cells per sample were targeted.