Single-nucleus RNA sequencing of healthy human adult lung from trachea, bronchi and bronchioles
Single-nucleus RNA sequencing of healthy human adult lung from trachea, bronchi and bronchioles
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. Tissue was shipped overnight in UW-Belzer's solution (Thermo Fisher, Cat#NC0952695) on ice and was processed for experiments or fixation within 24h. Normal, de-identified, donor human adult lungs not eligible for transplant were obtained from Gift of Life Michigan Donor Care Center. Specific tissue regions were dissected according to anatomic region and snap-frozen in liquid nitrogen for further downstream analysis. Preparation was performed as described previously in accordance with 10X Genomics' protocols  CG000505 Rev A for adult tissue). Briefly, snap-frozen tissue was minced into smaller fragments using a scalpel and then added to lysis buffer (Thermo Fisher Scientific, Cat#PI28324). Tissue was homogenized using a pellet pestle 15 times then incubated for 5 minutes in lysis buffer. The cell suspension was filtered through a 33 μm strainer. Suspension was centrifuged at 500g for 5 mins at 4°C. Supernatant was removed and the pellet was washed with PBS + 1% BSA, centrifuged at 500g for 5 mins at 4°C; pellet was washed and centrifuged again. An added debris removal column step was used for the adult tissue.  Permeabilization for ATAC-seq samples was performed by incubating the pellet in 0.1X lysis buffer and incubated for 2 minutes. The suspension was then centrifuged at 500g for 5 mins at 4°C; supernatant was removed and pellet was resuspended in diluted nuclei buffer. Immediately after tissue dissociation, single nucleus libraries were prepared on the Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Bundle, 16 rxns PN-1000283 was used with a target of 7500 cells.