Monoclonal anti flag m2 antibody produced in mouse

Sixty OD of cells at OD 2 were resuspended in 800 µL lysis buffer (20 mM Tris/HCl pH 8.0, 150 mM KCl, 1 mM MgCl₂, 1 mM DTT), and 800 µL 0.1 mm glass beads (BioSpecs). Cells were lysed at 30 Hz for 10 min in the Retsch MM200 at 4°C. The lysate was cleared twice for 10 min at full-speed at 4°C. An amount of 35 µL monoclonal anti-FLAG M2 antibody produced in mouse (Sigma-Aldrich) was added to the supernatant and incubated for 30 min at 4°C. An amount of 75 µL prewashed protein A sepharose (Sigma-Aldrich) was added for recovery of FLAG-antibody and incubated for 30 min. Beads were pelleted at 300g and washed five times with lysis buffer, resuspended in 500 µL lysis buffer and extracted with PCI. The aqueous layer was precipitated with ethanol and 1:30 3 M sodium acetate at pH 5.2 at −20°C overnight. The precipitated RNA was pelleted and washed with 500 µL 75% ethanol. The pellet was dried and solubilized in 15.5 µL water. DNA was degraded by DNase I (Thermo Scientific) as described in the manual in the presence of RNase inhibitor for 30 min at 37°C. The sample was diluted with 100 µL water and the RNA was extracted by PCI. The RNA was solubilized to an OD₂₆₀ of 1 and analyzed in the Bioanalyzer (Agilent) for quality and quantity estimation for RNA-seq. Protein samples were recovered from the organic layer by 10 vol methanol precipitation.