GSM5333796: RH30 cells - INPUT_rep2; Homo sapiens; RIP-Seq
For RIP from nuclear extracts, RH30 cells were resuspended in buffer A/NT1 (20mM TrisHCl pH8.0, 10mM NaCl, 3mM MgCl2, 0.1% NP40, 10% Glicerol, 0.2mM EDTA, 0.4mM PMSF, 1XPIC). In ice for 15 minutes and centrifuge at 2000 rpm for 5 minutes at 4°C to pellet the nuclei. The supernatant was collected as cytoplasmic extract. The pellet was rewashed with buffer A for 3 times. The pellet was resuspended in NT2/Wash buffer (50mM Tris pH7.4, 150mM NaCl, 1mM MgCl2, 0,5% NP40, 20mM EDTA, 1X PIC, 1X PMSF, 1 mM DTT), break with 7ml dounce and centrifuged at 14000 rpm for 30 minutes at 4°C. Protein A/G Magnetic beads (Thermo Fisher Scientific) were incubate with IgG, DDX5 (ab126730) antibody on rotating wheel O/N at 4°C while the nuclear extract was precleared with the beads. Trio RNA-Seq kit (Tecan Genomics, Redwood City, CA) has been used for library preparation following the manufacturer's instructions (library type: unstranded). RNA samples were quantified and quality tested by Agilent 2100 Bioanalyzer RNA assay (Agilent technologies, Santa Clara, CA) or Caliper (PerkinElmer, Waltham, MA). Final libraries were checked with both Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) and Agilent Bioanalyzer DNA assay or Caliper (PerkinElmer, Waltham, MA).