GSM6598198: Young, replicate 1, scATAC-seq; Mus musculus; ATAC-seq
Young (3 months old) and aged (28-29 months old) Pax7CreER/+;Rosa26TdTomato/+  female mice were obtained from a breeding colony at UM. All mice were fed normal chow ad libitum and housed on a 12:12 hour light-dark cycle under UM veterinary staff supervision. All procedures were approved by the University Committee on the Use and Care of Animals at UM and the Institutional Animal Care and Committee and were in accordance with the U.S. National Institute of Health (NIH). Mouse hindlimb muscles were extracted and placed into separate petri dishes containing ice-cold PBS. Using surgical scissors, muscle tissues were minced and placed into 20mL of digestion buffer (DMEM with Collagenase type II (0.2%) and Dispase II (2.5U/mL)) per mouse. Samples were placed on a shaker in a 37˚C incubator for 1.5 hours and mixed by pipette every 30 minutes. The enzymes were then inactivated by addition of 20mL 20% heat-inactivated fetal bovine serum (HI-FBS) in Ham's F10 media. The solution was passed through a 70um cell strainers, centrifuged, washed in PBS containing 3% BSA, and nuclei isolation was performed according to 10x Demonstrated Protocol CG000375 Revision B starting at step 1.1c. Nuclei from two age-matched mice were pooled prior to sorting, at step 1.1n, and 7-AAD+ nuclei were sorted on a Sony MA900 or MoFlo Astrios 3. Sorted nuclei were then permeabilized according to the same protocol. 10,000 permeabilized nuclei were loaded onto the 10x Genomics Chromium single cell controller and single nuclei were captured into nanoliter-scale gel bead-in-emulsions (GEMs). Single nuclei ATAC library constructions were performed using the ATAC-seq Next GEM kit (10x Genomics). All libraries were submitted for 51x51bp paired-end sequencing on a NovaSeq 6000 with 50,000 targeted reads per cell.