title: GSM1258126: mdx_young_tsa; Mus musculus; OTHER
library_construction_protocol: Fibro/adipogenic progenitors (FAPs) cells were isolated as Ter119-/CD45-/CD31-/α7integrin-/Sca-1+ cells. Hind limb muscles were minced and digested in HBSS (Gibco) containing 2 μg/mL Collagenase A (Roche), 2.4 U/mL Dispase I (Roche), 10 ng/mL DNase I (Roche), 0.4 mM CaCl2 and 5mM MgCl2 for 90 min at 37°C. Cells were stained with primary antibodies (10 ng/mL) CD31-eFluor450 (eBioscience), CD45-eFluor450 (eBioscience), TER-119-eFluor450 (eBioscience), Sca-1-FITC (eBioscience) and α7integrin-APC for 30 min on ice. Cells were finally washed and resuspended in HBSS containing 0.2% (w/v) BSA and 1% (v/v) Penicillin-Streptomycin. Flow cytometry analysis and cell sorting were performed on a DAKO-Cytomation MoFlo High Speed Sorter. The library for NA-seq was prepared as described in Gargiulo et al. (PMID: 19289091) with some modifications. 1e6 FAPs freshly isolated by FACS, were washed twice with ice‐cold PBS, and centrifuged for 5 min at 500g at 40°C. Cells were resuspended in 1 ml ice-cold homogenization buffer (10 mM Tris-Cl pH 7.4, 15 mM NaCl, 60 mM KCl, 5%sucrose, 1 mM EDTA, 0.1 mM EGTA, 0.1% NP-40, 0.15 mM spermine, 0.5mM spermidine, 1 mM DTT, 0.5 mM PMSF). The homogenate was transferred to a fresh tube and 400 μl of a sucrose "pad" solution (10 mM Tris‐Cl pH 7.4, 15 mM NaCl, 60 mM KCl, 10% sucrose, 1 mM EDTA, 0.1 mM EGTA, 0.15 mM spermine, 0.5 mM spermidine, 1 mM DTT, 0.5 mM PMSF) was added to the homogenate to form a layer underneath it. The nuclei were pelleted at 140g at 4°C for 20 min and gently resuspended in 100 μl of ice-cold reaction buffer (10 mM HEPES pH 7.5, 25 mM KCl, 5 mM MgCl2, 5% glycerol, 0.15 mM spermine, 0.5 mM spermidine, 1 mM DTT, 0.5 mM PMSF), incubated at 37°C for 1 min, and then treated with 1/10th v/v of NlaIII or HpaII (NEB, 50'000 units/ml) for 5 min. at 37°C. The reaction was stopped by adding 0.5M EDTA to reach a final concentration of 25mM. After purification the second enzymatic digestion was perfomed using Sau3aI (20 U) for 2h at 37°C and again purified. Libraries for sequencing on HiSeq2000 were prepared according to the Illumina standard protocol.
attributes.tag: GEO Accession
attributes.value: GSM1258126