Vcx130

ChIP assays were performed using the ChIP Assay Kit (Beyotime). Briefly, after crosslinking the chromatin with 1% formaldehyde at 37°C for 20 min and neutralizing with glycine for 5 min at room temperature, C2C12 myoblasts were washed with cold phosphate-buffered saline, scraped, and collected. Nuclear lysates were sonicated 15 times for 10 s at 10-s intervals on ice using a Sonics VCX 130 (Sonics, USA). The chromatin complex was immunoprecipitated at 4°C overnight with pCREB (Ser133) rabbit antibodies using phenol/chloroform. Real-time PCR was performed to analyze the DNA fragments. The primers were MyoD-cF (forward primer, 5’-CCCAGATGGAGAATGACCAAA-3’), MyoD-cR (reverse primer, 5’- AAGGCTACGGGACAATGAAAG-3’) and MyoG-cF (forward primer, 5’-TCAAAGAAGCTGTAGAAACCCAA-3’), MyoG-cR (reverse primer, 5’-TCAGCAG CACCTTAAACCATAC-3’). According to the ChIP analysis manual (Thermofisher scientific), Relative enrichment is calculated as the amount of amplified DNA normalized to input and relative to values obtained after normal IgG immunoprecipitation.

The particle size and distribution were determined by laser diffraction. For the calculation of the size distribution, a refractive index of 1.5 was used and the imaginary component of the refractive index was 0.5. The particle size was reported as a volume mean diameter, marked as D(4,3), and the size distribution with d10, d50 and d90 data. The measurements were carried out in wet dispersion in 100 mL of cyclohexane containing 0.1 m/m% soy lecithin with a Malvern Mastersizer 2000 (Malvern Instruments, Malvern, UK) using the SM dispersion unit with a stirring rate of 2000 rpm. A sample of 10 mg was taken in 1 mL of cyclohexane solutions containing 0.1 m/m% soy lecithin and sonicated for 40 s at 30% of power with a 6 mm probe using a Sonics VCX 130 (Sonics & Materials, Newtown, CT, USA), after which the obtained suspension was loaded onto the dispersion unit for measurement.

The ChIP assay was performed using a ChIP Assay Kit (Millipore) according to the manufacturer’s instructions. Briefly, dCLN CD8⁺ T cells were isolated and fixed in 1% formaldehyde for 10 min at room temperature. Fixed cells were washed and then lysed in ChIP lysis buffer. The whole-cell extracts were then sonicated for 10 cycles of 10 s on /20 s off and 50% AMPL with Sonics VCX130 (Sonics & Materials, Inc, Newtown). Antibodies directed against p53 (ProteinTech, 10442-1-AP, 1 μg) or rabbit IgG (ProteinTech, Cat# B900610, 1 μg) were used. The precipitated DNA was subjected to PCR amplification. The primer sequences used in ChIP assays:

ChIP was performed using a ChIP Assay kit (Millipore, Billerica, MA) according to the manufacturer's instructions. Briefly, 2 × 10⁶ (for AcH4, H3K4me3, H3K9me3 and H3K27me3) or 5 × 10⁶ (for STAT6, Sp1 RNA pol II and HDAC2) macrophages were stimulated with 20 ng ml⁻¹ IL-4 for the indicated time, washed in PBS, and fixed in 1% formaldehyde for 10 min at room temperature. Fixed cells were harvested, lysed and sonicated for 10 cycles of 10 s on/20 s off and 50% AMPL with Sonics VCX130 (Sonics & Materials, Inc, Newtown). For AcH4 ChIP, sodium butyrate(20 mM, 19–137, Millipore) was added to all of the solutions to preserve histone acetylation. Antibodies directed against STAT6 (sc-621X; 5 μg per 1 mg total protein) and Sp1 (sc-59X; 5 μg per 1 mg total protein) were obtained from Santa Cruz, and antibodies directed against pol II (05–623; 10 μl per 1 mg total protein), AcH4(06–866; 10 μl per 1 mg total protein), H3K4me3 (07–473; 5 μl per 1 mg total protein), H3K9me3 (05–1242; 10 μl per 1 mg total protein), H3K27me3 (07–449; 5 μl per 1 mg total protein) and HDAC2 (17–10237; 5 μl per 1 mg total protein) were obtained from Millipore. The precipitated DNA was subjected to PCR amplification. The primer sequences used in ChIP assay are described in Supplementary Table 3.

Ultrasonication at two different amplitudes: 40% and 60% using Sonics VCX 130 (Sonics & Materials, Inc., Newton, CT, USA) was conducted for redispersing nanogel powder and creating a water-in-oil (w/o) mini-emulsion, respectively. Purification of bare nanogels and NG/miRNA was achieved by a dialysis method following lyophilization in a freeze-dryer (ALPHA 1-2 LDplus, CHRIST) under 0.035 mbar at −50 °C. Deionized water (DI water) was produced using a reverse osmosis system (conductivity < 2 μS/cm).