GSM6454670: Nucleolin binding targets_3, normal conditions; Mus musculus; RNA-Seq
The cells were washed with PBS, and irradiated with 0.15 mJ cm-2 and 365 nm ultraviolet (UV) light in a UV crosslinker to crosslink the labeled RNA to RNA-binding proteins. After isolation of nucleoli from cell lysates, nucleolar extracts were treated with 1 U ml-1 RNase T1 and NCL proteins were immunoprecipitated with anti-NCL antibodies and Protein A/G sepharose beads. The immunoprecipitates were further digested with 100 U ml-1 RNase T1 and the beads were washed in lysis buffer and suspended in one bead volume of dephosphorylation buffer. RNAs were dephosphorylated and radioactively labeled with [γ -32P]-ATP. The protein-RNA complexes were separated by SDS-PAGE and visualized by autoradiography. The radioactive bands migrated as a 110 kDa were eluted from the gel and all proteins in eluted bands were removed by digestion with 0.2 mg ml-1 proteinase K. The RNAs were isolated by acidic phenol/chloroform extraction and ethanol precipitation. cDNA libraries were prepared for sequencing using standard Illumina protocols