After cell lysis in 3 mL hypotonic lysis buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 2 mM EDTA, 0.5% (v/v) Triton X-100, Halt Protease Inhibitor Cocktail), and removal of cell debris by centrifugation, the supernatant was adjusted to 160 mM NaCl and incubated with 30 U RNase I (Ambion) and 15 U Turbo DNase (Ambion) at 37 °C for 7.5 minutes. 160 μL Dynabeads Protein G were incubated with 18 μg of mouse anti-FLAG M2 antibody (Sigma Aldrich), washed and resuspended in 1 mL hypotonic lysis buffer and incubated with the cell lysate at 4 °C for 1.5 hours. The beads were then washed three times with IP-buffer (50 mM HEPES-NaOH pH 7.5, 300 mM KCl, 0.05% (v/v) NP-40, Halt Protease Inhibitor Cocktail). Illumina TruSeq Stranded mRNA.
GEO Accession
GSM2298849