C18 bond elut omix 100 μl pipette tips

Cell lysates were prepared using the FASP method^{49 (link)}. Briefly, 8 volumes of 8 M urea was added to each of the protein-containing supernatants and loaded onto an Amicon 0.5 mL 30,000 molecular-weight-cutoff (MWCO) centrifugal ultrafiltration unit (EMD Millipore). Proteins were washed with 8 M urea, reduced, alkylated with iodoacetamide, washed again with 1 M urea, and then digested with Lys-C (Wako) [40:1 (w/w) protein/enzyme ratio] at 37 °C overnight to generate peptides. The resulting peptides were small enough to pass through the MWCO filter and collected after centrifugation. Lys-C was quenched using 10% trifluoroacetic acid (TFA) to a final concentration of 0.5%. Peptides were desalted using C18 Bond Elut OMIX 100-μL pipette tips (Agilent) and eluted with 0.1% TFA in 70% acetonitrile. Peptides were dried in a SpeedVac and reconstituted in 5% acetonitrile and 0.2% formic acid.

One μL of whole blood from each mouse was lysed using 4 μL of 8 M urea and 10 mM tris-HCl (pH 7.5). Samples were bath sonicated for 3 min to shear chromatin. Next, 7.5 μL of 50 mM ammonium bicarbonate was added followed by 1.25 μL of 10 mM dithiothreitol. Samples were incubated at room temperature for 1 h to allow for reduction of cysteines, then 1.25 μL of iodoacetamide was added and incubated without light for 40 min to acetylate cysteines. Samples were diluted with 40 μL of 50 mM ammonium bicarbonate prior to digestion with 1 μg of Lys-C. Samples were digested at 37 °C overnight and quenched with 3.25 μL TFA. Peptides were desalted using C18 Bond Elut OMIX 100 μL pipette tips (Agilent) and eluted with 0.1% TFA in 70% acetonitrile. Peptides were dried in a SpeedVac and reconstituted in 5% acetonitrile and 0.2% formic acid.