GSM9531576: Lung, KO-2; Mus musculus; RNA-Seq
Lung tissue from 10-month-old wild-type and Parp7–/– mice (n = 2 per genotype) were isolated at death, snap-frozen in liquid nitrogen, and stored at -80°C. Nuclei were prepared from frozen tissue by cutting lung into ~5 mm pieces, injecting lung pieces with 1 mL Nuclei EZ Lysis Buffer (#NUC101; Sigma-Aldrich) supplemented with 1x protease inhibitor (589279100; Roche), 200 U/mL Protector RNase Inhibitor (#3335399001; Sigma-Aldrich), and 200 U/mL SUPERaseIN (AM2696; ThermoFisher). Lung tissue was further minced with an additional 1 mL of supplemented Nuclei EZ Lysis Buffer and then transferred to a GentleMACS C tube. GentleMACS program m_lung_01.01 was run followed by m_lung_02.01 for 20 seconds. Lysate was briefly spun down at 750 xg for 2 min. to spin down the foam. Lysate was passed through a 40 μm strainer and rinsed with PBS supplemented with 0.04% BSA (AM2616; ThermoFisher), 200 U/mL Protector RNase Inhibitor, and 200 U/mL SUPERaseIN. Nuclei were spun down at 1000 xg for 5 min. at 4°C followed by another wash. Nuclei were resuspended in 1 mL supplemented PBS and passed through a 40 μm Flowmi Cell Strainer (BAH136800040) twice. Nuclei were counted with a hemocytometer and diluted to 700-1200 nuclei/μL. Single-nuclei RNA-seq libraries were prepared using the Chromium Single Cell 5' Reagent Kits (10x Genomics) according to the manufacturer's protocol.