GSM6304274: SRSF2 PAR-CLIP Seq in SRSF2(P95H) K562 cell rep2; Homo sapiens; RIP-Seq
GSM6304274
The cells were lysed, digested with 1 U/μl RNase T1 at 22 °C for 8 min, and immunoprecipitated with anti-Flag M2 magnetic beads (Sigma). The protein-RNA-bead complex was digested with 10 U/ μl RNase T1 again at 22°C for 8 min and incubated with 0.5 U/μl Alkaline Phosphatase Calf Intestinal (CIP, NEB) for 10 min at 37°C. The beads were washed and then incubated with 0.5 U/μl T4 Polynucleotide Kinase (NEB) for 15 min at 37°C. After washing, one-sixth of the beads were resuspended in LDS loading buffer (Invitrogen), boiled at 95°C for 10 min, and the mixture was resolved by SDS-PAGE to detect the immunoprecipitation efficiency. One-sixth of the beads were labeled with biotin using the RNA 3′ End Biotinylation kit (Thermo), following the manufacturer's instructions for PAR-CLIP-biotin chemiluminescent nucleic acid detection. The rest of the beads were also boiled and the mixture electrophoresed through a NuPAGE Bis-Tris protein gel. Parts containing target protein-RNA complexes were cut from the gel according to the protein-RNA-biotin signal. The protein-bound RNA in the gel pieces was recovered by D-Tube™ Dialyzer Midi (Merck-Millipore), digested with proteinase K (Roche), and extracted with phenol-chloroform. Library preparation was performed using the Takara SMARTer smRNA-seq kit (PAR-CLIP in HeLa cells) or the NEBNext® Multiplex Small RNA Library Prep kit (PAR-CLIP in K562 cells) for Illumina, following the manufacturer's instructions.