GSM8572003: MelSTR cells, input control 3; Homo sapiens; RIP-Seq
Cells were lysed in hypotonic gentle lysisbuffer (10 mM Tris-Hcl, 10 mM NaCL, 2 mM EDTA and 0.5% triton) and centrifuged for 10 min at maximum speed at 4°C. Supernatant was diluted in 1xNet buffer (50 mM Tris-Hcl, 150 mM NaCl, 0.1% NP40, 1 mM EDTA and RNAse inhibitor (Promega, #N2111) and incubated for 2h at 4°C on a wheel with 50 ul Dynabeads A previously coupled to 3 ug eIF2A or IgG antibody, respectively. Afterwards beads were washed 5 times with 500 ul 1xNet buffer. RNA was extracted from the remaining beads using Trizol. For input samples, RNA was directly extracted with Trizol after extract collection. RNA immunoprecipitations (RIP) were performed under native conditions using an eIF2A antibody (abcam #ab169528), an IgG rabbit control antibody and Dynabeads A (invitrogen, #10002D).. Libraries were prepared with poly-A selection for 50bp Illumina sequencing