GSM6598197: Aged, replicate 2, Hi-C; Mus musculus; Hi-C
Young (4 months) and aged (24-26 months) Pax7CreER/+;Rosa26TdTomato/+ female mice were obtained from a breeding colony at the University of Michigan (UM). All mice were fed normal chow ad libitum and housed on a 12:12 hr 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 were in accordance with the U.S. National Institute of Health (NIH). In brief, hind limb muscles were harvested from mice using sterile surgical tools. Muscle tissue was minced and digested in 20 mL of a digestion solution (2.5 U/mL Dispase II and 0.2% [~5500 U/mL] Collagenase Type II in Dulbecco's modified Eagle medium [DMEM]). Samples were incubated at 37°C for 60 min. Once the digestion was complete, 20 mL of F10 media containing 20% heat inactivated FBS was added into each sample to inactivate enzyme activity. The solution was then filtered through a 70 µm cell strainer into a new 50 mL conical tube and centrifuged at 350× g for 5 min. Subsequently, the protocol for crosslinking low input mammalian cells was followed, as written in the Arima-HiC kit workflow provided by Arima Genomics Inc (San Diego, CA). Briefly, the pellets were re-suspended in 1X PBS and 37% formaldehyde was added to obtain a final concentration of 2% formaldehyde. The samples were inverted and incubated at RT for 10 mins. Arima-Hi-C Stop Solution 1 was added to samples, and they incubated for an additional 5 mins. The samples were then placed on ice to incubate for 15 mins. Cells were pelleted by centrifugation for 5 min at 500 × g, the supernatant was discarded, and cells were resuspended in staining media (2% heat-inactivated FBS, 2 mM EDTA in Hank's buffered salt solution) and antibody cocktail containing Sca-1:APC (1:400), CD45:APC (1:400), CD11b:APC (1:400), Ter119:APC (1:400), CD29/β1-integrin:PE (1:200), and CD184/CXCR-4: BIOTIN (1:100) and incubated for 30 min on ice in the dark. Cells and antibodies were diluted in 3 mL of staining solution, centrifuged at 350× g for 5 min, and supernatants discarded. Pellets were re-suspended in 200 µL staining solution containing PECy7:STREPTAVIDIN (1:100) and incubated on ice for 20 min in the dark. Again, samples were diluted in 3 mL staining solution, centrifuged, supernatants discarded, and pellets re-suspended in 200 μL staining buffer. Samples were filtered through 70 μm cell strainers before the FACS. In situ Hi-C was performed using the Arima-HiC Protocol. Approximately 75,000-100,000 sorted muscle cells were crosslinked, permeabilized and chromatin digested as specified by the manufacturer in biological replicates. Restriction fragment ends were then labeled with biotinylated nucleotides and proximal DNA ligated, followed by reversal of cross-links. The DNA was then sheared using Diagenode's Bioruptor sonicator for 4 cycles of 15 sec on, 90 sec off, then briefly centrifuged. An additional 4 cycles of sonication were performed after centrifugation. The resultant DNA was size-selected using AMPure XP beads followed by verification with the Arima Quality Control 2 assay. Biotinylated junctions were then magnetically isolated with streptavidin coated beads, followed by end-repair, A-tailing, sequencing adaptor ligation and PCR amplification. The resultant libraries were then size-selected using AMPure XP beads and sequenced with 150 bp paired-end reads at a depth of 750 million reads per library on an Illumina NovaSeq flow cell.