Single-nucleus ATAC sequencing of human fetal lung from 4 anatomic regions
Single-nucleus ATAC 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. 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 nuclei isolation. Preparation was performed as described previously in accordance with 10X Genomics' protocols (CG000375 Rev B for fetal 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 1x nuclei buffer (10X) and submitted to the University of Michigan Advanced Genomics Core. Single-cell multiome libraries were generated using the 10x Genomics Chromium Single Cell Multiome ATAC + Gene Expression platform according to the manufacturer's protocol. Briefly, nuclei were isolated from tissue samples and loaded onto the Chromium Controller for partitioning into Gel Bead-In-Emulsions (GEMs). Within each GEM, individual nuclei were barcoded with a unique cell barcode. Accessible chromatin was simultaneously profiled by Tn5 transposase-mediated tagmentation, while polyadenylated RNA molecules were captured and reverse transcribed to generate barcoded cDNA. Following GEM recovery, separate gene expression and chromatin accessibility libraries were constructed according to the Chromium Single Cell Multiome workflow. Gene expression libraries contained cell barcodes and unique molecular identifiers (UMIs) linked to cDNA molecules, whereas ATAC libraries contained cell barcodes linked to accessible chromatin fragments. Libraries were amplified, purified, quality controlled, and sequenced on an Illumina platform using paired-end sequencing.