Magnetic proteina proteing beads

To generate recombinant p53 we in vitro transcribed/translated human p53 with a c-terminal HA tag using a rabbit reticulocyte system (Promega). To generate fragmented genomic DNA we tagmented 50 ng of human genomic DNA from MCF7 cells using the MuSeq kit (Thermo) and amplified it using PCR and custom adaptor primers for 8 cycles. DNA was cleaned up on SPRI beads (Aline Biosciences) and quantified. At room temperature 20 ng of DNA and recombinant p53 (0.1 μM final) were combined in a binding buffer (10 mM TRIS, 5 mM MgCl2, 10% glycerol, 1 mM DTT) and incubated at room temperature for 30 min. The mixture was diluted 2-fold (to 20 μl) and 1.5 μl of anti-HA antibody was added (Rockland) and the sample incubated at 4C overnight with shaking. A 1:1 mixture of magnetic proteinA/proteinG beads was added (Sigma) and incubated at 4C for 1 h with shaking. The beads were then washed 3x with washing buffer (10 mM Tris, 5 mM HCL, 0.1% triton, 150 mM NaCl) and DNA eluted with elution buffer (1%SDS, 100 mM Na2CO3) at 37C for 15 min. Samples were cleaned up, and adaptors and barcodes added by PCR. Reads (> 30 M) were trimmed to remove adaptors with cutadapt [38 ], aligned to the genome with Bowtie, and analyzed with Matlab.

RNA immunoprecipitation (RIP) was performed using a MagnaRIP Kit (Millipore) as described previously (Lu et al., 2016 (link)). Briefly, Cells were harvested and lyzed with RIP lysis buffer (10 mM HEPES, pH 7.0, 100 mM KCl, 5 mM MgCl2, 0.5% NP-40, 1 mM DTT) for 20 min on ice. After centrifuge, the whole cell lysates were incubated at 4°C overnight with magnetic protein A-protein G beads (Sigma-Aldrich) coupled with isotype IgG control or CPEB4 antibody to obtain RNA-protein immunocomplexes. Beads were washed three times with washing buffer, and incubated with proteinase K buffer for 45 min at constant 55°C, which was followed by RNA isolation from the immunoprecipitates according to the manufacturer’s instructions. cDNA was reversely transcripted by First-strand cDNA Synthesis System (Thermo Scientific, K1621). qRT-PCR was performed by amplifying a 300-bp region in the 3′ UTR of each transcript.