GSM1419784: CLIP_BMDM_KO3; Mus musculus; RIP-Seq
BMDMs were scrapped off the plates, flash-frozen and stored at -80C. Frozen cells were thawed and lysed in 1mL PXL lysis buffer (1x PBS, 0.1% SDS, 0.5% Na-DOC, 0.5% NP-40) with complete protease inhibitor. After lysing on ice with occasional vortexing, the cells were subjected to DNase treatment with RQ1 RNAse-free DNase (Promega) for 5 minutes at 37°C shaking in a Thermomixer. RNAse treatment performed with high (1:100) or low (1:10,000) RNAse A solutions and incubated for 5 minutes shaking at 37°C. Lysates were spun down (14,000RPM, 30minutes at 4°C) and the supernatant transferred to a new tube. Protein A beads were pre-loaded with rabbit anti-mouse IgG bridging antibody (Jackson ImmunoResearch, at 2.3 mg/mL), incubated for 35 minutes at room temperature. Beads were washed (in 0.02% Tween) and then loaded with 3 uL Ago antibody (2A8 ascites provided by Dr. Zissimos Mourelatos- (Nelson et al., 2007)) per 400 uL of beads and rotated for 3 hours at 4°C. Cleared lysates were incubated with primary antibody-loaded beads for 2 hours at 4°C and washed in a series of washes: 1) 2x 1x PXL buffer; 2) 2x 5x PXL (5xPBS 0.1 % SDS, 0.5% Na-DOC, 0.5% NP-40); 3) 2x with high stringency buffer (15mM Tris-HCl, pH 7.5, 5mM EDTA, 2.5mM EGTA, 1% Triton X-100, 1% Na-DOC, 0.1% SDS, 120mM NaCl, 25mM KCl); 4) 1x with high salt buffer (15mM Tris-HCl, pH 7.5, 5mM EDTA, 2.5mM EGTA, 1% Triton X-100, 1% Na-DOC, 0.1% SDS, 1M NaCl); 5) 2x with low salt buffer (15mM Tris-HCl, pH 7.5, 5mM EDTA); 6) 2x NT-2 buffer (50mM Tris-HCl pH 7.4, 150mM NaCl, 1mM MgCl2, 0.05% NP-40); 7) 2x PNK buffer (50mM Tris-HCl, pH 7.5, 10mM MgCl2, 0.5% NP-40). Dephosphorylation of the 5’ phosphate was performed with calf intestinal phosphatase (Roche) with RNAsin Plus RNAse Inhibitor (Promega) at 37°C for 20 minutes, shaking at 1000rpm for 15s every 2 min. Beads were then washed 1) 1x PNK buffer 2) 2x PNK buffer supplemented with 20mM EGTA 3) 2x PNK buffer. The puromycin blocked linker was radio-labeled with T4 PNK (New England Biolabs) and 32P-γ-ATP for 30 min at 37°C. Radiolabeled linker was spun through a G-25 column to remove free-ATP. Labeled 3’RNA linker was ligated to the 3’ end of the RNA with T4 RNA ligase (Fermentas) incubated at 16°C for 1 hour, shaking at 1000rpm for 15s every 4 min. After 1 hour, 80 pmole of cold L32 RNA linker with 5’ phosophate was added. Sampels were incubated overnight and washed 3x with PNK buffer. 5’ ends were re-phosphorylated with with T4 PNK for 20 min at 37°C shaking at 1000rpm for 15s every 4 min. Protein:RNA complexes were eluted off the beads in NuPAGE loading buffer (Invitrogen) at 70°C for 10 min shaking at 1000rpm. Supernatants were run on Novex NuPAGE 8% Bis-Tris gels (Invitrogen) in MOPS running buffer (Invitrogen) for 2hr at 175V and transferred onto Protran BA85 nitrocellulose (Whatman) and exposed to Biomax MR film (Kodak). Regions that corresponded to Ago:mRNA and Ago:miRNA complexes were excised from the membrane, diced and treated with proteinase K (4mg/mL Roche) for 20 min at 37°C shaking at 1100rpm. RNA was then extracted via phenol-chloroform extraction and ethanol precipitation. 5’ linkers with a degenerate nucleotide end were ligated to the extracted RNA with T4 RNA ligase (Fermentas) at 16°C for 5 hours. The ligated reaction was then subjected to DNase treatement with RQ1 DNase (Promega) for 20min at 37°C and extracted with phenol-chloroform and subjected to ethanol precipitation. Precipitated RNA was reverse transcribed with Superscript III (Invitrogen) and PCR amplified with Accuprime Pfx Supermix (Invitrogen) for 20-35 cycles. PCR products were run on a 10% denaturing polacrylamide gel and visualized with SYBR Gold (Molecular Probes) staining. 60-100nt products were excised from the lowest cycle number with visual product and gel extracted. Additional PCR amplification followed by gel extraction was performed as described with fusion primers to provide the platform for Illumina sequencing. 10-30uL of 10nM DNA was submitted for sequencing on Illumina HiSeq.
GEO Accession
GSM1419784