The protocol was adapted from the RIP–ChIP protocol described previously45 (link). Briefly, protein A Sepharose beads (Sigma) were coated with 3 µg of U1 snRNP, SF3B1, SRSF1, U2AF2 or mouse IgG antibody (Santa Cruz), followed by incubation with 2 mg of Hep3B or SNU398 total cell lysates overnight. The RNA–protein–bead complexes were washed once with NT2 crowders (25 mg Ficoll PM400 (GE Healthcare), 75 mg Ficoll PM70 (GE Healthcare) and 2.5 mg dextran sulfate (Fluka) in 10 ml of NT2 buffer) and five times with NT2 buffer (50 mM Tris pH 7.0, 150 mM NaCl, 1 mM MgCl2 and 0.05% (v/v) NP-40). Protein–RNA complexes were collected in 100 μl of NET2 buffer (1 mM DTT, 16.7 mM EDTA, 200 U RNaseOUT (Thermo Fisher) and 100 U SUPERase In (Ambion) in 1× NT2 crowder), supplemented with 100 μl of 2× SDS–TE (100 mM Tris pH 7.5, 10 mM EDTA pH 8.0 and 1% SDS). RNA was isolated using TRIzol reagent and subsequently purified with phenol:chloroform:isoamyl alcohol (25:24:1) and chloroform:isoamyl alcohol (24:1).
Buffer Cell ChipChloroform Dextran sulfate EdtaFicoll Igg antibody Isoamyl alcohol Mgcl2 Mouse Nacl Np 40Phenol Protein Protein a sepharose TrisTrizol U1 snrnp U2af2
Corresponding Organization :
Other organizations :
National University Cancer Institute, Singapore, National University of Singapore, King Abdullah University of Science and Technology, Agency for Science, Technology and Research, Nanyang Technological University, National University Health System, National Cancer Centre Singapore, Genome Institute of Singapore, University of Malaya, National Cancer Institute of Thailand, Singapore General Hospital, Icahn School of Medicine at Mount Sinai
Antibody used for immunoprecipitation (U1 snRNP, SF3B1, SRSF1, U2AF2 or mouse IgG antibody)
dependent variables
RNA isolated from the protein-RNA complexes
control variables
Total cell lysates from Hep3B or SNU398 cell lines
NT2 buffer composition (50 mM Tris pH 7.0, 150 mM NaCl, 1 mM MgCl2, 0.05% (v/v) NP-40)
NET2 buffer composition (1 mM DTT, 16.7 mM EDTA, 200 U RNaseOUT, 100 U SUPERase In, 1× NT2 crowder)
2× SDS–TE buffer composition (100 mM Tris pH 7.5, 10 mM EDTA pH 8.0, 1% SDS)
controls
Positive control: Immunoprecipitation with specific antibodies (U1 snRNP, SF3B1, SRSF1, U2AF2)
Negative control: Immunoprecipitation with mouse IgG antibody
Annotations
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