GSM7852464: Alveolar Epithelial Type 2 cells from normal donor 1, RNA-seq; Homo sapiens; RNA-Seq
Lung parenchyma was macrodissected to a depth of > 2mm in order to isolate alveolar tissue. The visceral pleura was removed using forceps, and alveolar tissue was then minced to fine pieces with scissors. Tissue was then washed in 50:50 DMEM/F12 media, which is a 50:50 mixture of DME-F12 (#D64421, Sigma) and DMEM High Glucose (#21063, GIBCO) supplemented with 1% penicillin/streptomycin (), 0.1% Gentamycin (#G1272, Sigma), and 1% Fungizone (A2411, Sigma). Following overnight incubation, the tissue was again minced briefly using a razor blade and resuspended in HBSS media without Ca2+ or Mg2+ supplemented with 13U/mL of elastase (#LS002280, Worthington) for 30min at 37C. Cells were filtered through a gauze mesh and then through a 100um filter, then pelleted and resuspended in 50:50 DMEM/F12 media and applied to a Percoll density gradient centrifugation, followed by IgG panning (#I4506, Sigma). Magnetic-activated sorting with Epcam+ beads (Miltenyi Biotec #130-061-101) and FcR blocking (Biotec #130-059-901) was then performed, followed by separation using LS columns (Miltenyi Biotec #130-042-401). Following separation, Cells were counted using trypan blue to measure viability. After counting, cells were fixed in 4% paraformaldehyde (PFA) for 10 minutes, washed with Phosphate Buffered Saline (1xPBS), resuspended in PBS for each cytospin. Cytospins were prepared using EZ single cytofunnels (#1439045, Thermo Fisher Scientific) and a Shandon CytoSpin III cytocentrifuge (GMI, Ramsey, Minnesota). Cytospin slides were stored at -20°C until use. For RNA, 6 million AT2 cells were frozen into a pellet and stored at -80C. RNA was extracted using the Aurum Total RNA Mini kit (# 7326820, BioRad, Hercules CA) according to manufacturer guidelines. 2 ug of total RNA then underwent bulk RNA library preparation with PolyA selection performed by Genewiz (now Azenta, Burlington MA). Samples were barcoded using Illumina adapters to allow for multiplexed sequencing. For ATAC, between 5-50 K of freshly isolated AT2 cells were placed in 0.1 to 0.25 mL of ATAC lysis buffer (AM1) and immediately processed for ATAC library generation using the Active Motif ATACseq kit (#53150, Carlsbad CA) according to manufacturer guidelines. Nextera i7 dual indexing primers were used for library construction as listed in Table 1. Integration of indexing primers was performed with the Tn5 enzymes listed in Table 1. The Benchmark Scientific H5000-H MultiTherm shaker was used to ensure uniformity in library construction. The Agilent 4200 Tape Station system was used for assessment of ATAC fragment quality throughout library construction Bulk RNA paired end sequencing and bulk AT2 ATAC paired end sequencing was performed at Genewiz, using an Illumina HiSeq at 2x150 bp. RNAseq was performed using single index chemistry, whereas ATACseq was performed using dual-index chemistry.