Cells were lysed at 4 °C in RIPA buffer supplemented with protease inhibitors (Roche, 4693159001). Protein concentrations were measured using a BCA Assay Kit (Thermo Fisher, 23225), and 30 μg of total protein was resolved on precast 4–12% Bis-Tris gels (Invitrogen, NP0321BOX) and...
a, UMAP plots of benign breast epithelial cells and BC cells for all snRNA (left) and snATAC (right) samples. Tumor cells colored by PAM50 subtype.b, Heatmap of top 15 DEGs in snRNA-seq data from benign breast duct cells. A subset of genes from each benign cell type is highlighted ...
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Cells were incubated in buffer A (10 mM Tris, 1 mM KCl, 1.5 mM MgCl2) for 10 min on ice then dounce homogenized with 10 strokes. The mixture was centrifuged, and the pellet was resuspended in buffer B (50 mM Tris-HCl, 10 mM EDTA, 1% SDS) and kept for 10 min on ice. DNA was...
Peptide samples were dissolved in 10.8 μL 0.05% TFA & 2% ACN and injected 10 μL into the UHPLC coupled with a Q-Exactive HF-X mass spectrometer (Thermo Fisher Scientific). The mobile phase buffer consisted of 0.1% formic acid in HPLC grade water (buffer A) with an eluting buff...
All lyophilized samples were redissolved in 0.1% formic acid (buffer A) at a concentration of 5 mg/mL before MS analysis. All peptide mixtures were analyzed on a reverse phase C18 capillary LC column from Michrom Bioresources (100 μm x 150 mm, 3 μm, 0.5 μL/min). The elution grad...
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Briefly, in the presence of 100 nM E1 (Boston Biochem), 500 nM UbcH5c (Boston Biochem), 10 μM HA-ubiquitin (Boston Biochem), 5 mM ATP, and 2 μl of 10 × ubiquitination reaction buffer (500 mM Tris-HCl, pH 7.5, 50 mM MgCl2, 10 mM DTT), reactions were assembled with ...
Plates of 3T3-L1 cells at interval stages of adipogenesis were washed twice with cold PBS, lysed in TNET/OG lysis buffer, incubated on ice for 15 min, and then centrifuged for 10 min at 4 °C in a Beckman Microfuge at 14,000 rpm. Protein determinations were made on the post-nuclear ...
The isolated DNA was dissolved in 25 μl of EB buffer (Qiagen) and the quality quantity was detected by the Nanodrop ND 1000 spectrophotometer (Thermo Fisher Scientific, USA), and stored at -20 °C. To construct the Library, DNA was diluted to 10 ng/μl in a Li et al. ...