GSM8023967: Ptf1aERT;K*;Pdx1f/f, 10 weeks, replicate 1; Mus musculus; ATAC-seq
Frozen tissue was pulverized by using the CP02 pulverizer (Covaris). Pulver was washed with PBS containing 0.04% BSA and 1X protease inhibitor and centrifuged (5 min, 500 g, 4 °C). Supernatant was discarded, pellet was resuspended in 0.1 U/μl DNase solution (EN0521, Thermo Fisher Scientific) and incubated on ice for 5 minutes. Tissue was washed twice with PBS containing 0.04% BSA and 1X protease inhibitor. After the last centrifugation step (5 min, 500 g, 4 °C), supernatant was discarded, pellet was resuspended in freshly prepared lysis buffer (10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 0.1% Nonident P40, 0.01% Digitonin (BN2006, Thermo Fisher Scientific), 1% BSA and 1X protease inhibitor) and incubated on ice for 10 minutes. Nuclei were isolated by using a 2 ml Dounce homogenizer. Sample was filtrated through a 70 μm cell strainer, followed by centrifugation (1 min, 100 g, 4 °C). Supernatant was transferred to a new tube and washed with wash buffer (10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 1% BSA and 1X protease inhibitor) followed by centrifugation (5 min, 500 g, 4°C) for three times. Before the final washing step, suspension was filtered through a 40 μm Flowmi cell strainer. Nuclei were resuspended in Nuclei Buffer (10X Genomics). Library preparation and sequencing was performed at the Advanced Genomics Core of the University of Michigan. Single nucleus suspensions were counted on the LUNA Fx7 Automated Cell Counter (Logos Biosystems) and snATAC libraries were generated using the 10x Genomics Chromium instrument following the manufacturer's protocol. Final library quality was assessed using the LabChip GX (PerkinElmer).