GSM4852296: Empty Vector, no crosslink, replicate 2; Mus musculus; OTHER
Not-infected (NI), Empty Vector-infected (EV) and H-Ras-infected (Ras) PMK were cross-linked on ice at 0.15 J cm−2 with UV light at 254 nM. Immediately after irradiation, cells were lysed in 1 mL of lysis buffer (50 mM Tris-HCl, pH 7.4; 100 mM NaCl; 1% NP-40; 0.1% SDS; 0.5% sodium deoxycholate). Cell extracts were sonicated using a bioruptor (Digenode) for 10 cycles of 30 seconds, level L at 4ºC, then they cleared by centrifugation at 10'000 rpm for 10 min at 4°C. RNA was then partially digested by adding 10 μl of 1:200 dilution of RNase I (Ambion, AM2295), as well as 2 μl of Turbo DNase (Ambion, AM 2238) and incubating for 3 min at 37°C under shaking at 1100 rpm. CSDE1 protein was then captured by incubation with an affinity purified CSDE1 antibody conjugated to protein A Dynabeads for 4 h, 4°C, gentle rotation. Beads were then washed 2x with 900 μl high-salt buffer (50 mM Tris-HCl, pH 7.4; 1 M NaCl; 1 mM EDTA; 1% NP-40; 0.1% SDS; 0.5% sodium deoxycholate), 1x with 900 μl PNK wash buffer (20 mM Tris-HCl, pH 7.4; 10 mM MgCl2; 0.2% Tween-20) and resuspended in 40 μl of PNK mix (30 μl water; 8 μl 5x PNK pH 6.5 buffer [350 mM Tris-HCl, pH 6.5; 50 mM MgCl2; 5 mM dithiothreitol]; 1 μl PNK enzyme; 0.5 μl RNasin; 0.5 ul of FastAP alkaline phosphatase) and incubated 40 min at 37°C 1100 rpm. Beads were washed 1x with 900 μl of ligation buffer (50 mM Tris-HCl, pH 7.5; 10 mM MgCl2) and resuspended in 25 μl of ligation mix (6.8 μl water; 2.5 μl 10X ligation buffer; 0.8 ul 100% DMSO; 2.5 ul T4 RNA ligase I – high concentration [NEB]; 0.4 μl RNasin; 0.5 ul PNK enzyme; 2.5 μl pre-adenylated adaptor L3-App [1 uM] conjugated to an infrared-dye [IRdye-800CW-DBCO (LICOR, cat# 929-50000)]; 9 ul 50% PEG8000). Ligation was performed at room temperature for 75 minute flicking the tubes every 10 min. Beads were washed 2x with high-salt buffer, 1x with PNK wash buffer and resuspended in 20 ul of loading buffer and run on a 4-12% NuPAGE Bis-Tris gel (Invitrogen) in MOPS buffer according to the manufacturer's instructions. Protein-RNA complexes were transferred to a nitrocellulose membrane. Protein-infrared-RNA complexes were visualized at Odissey machine setting 1.5 intensity for both 700 nm and 800 nm channel. RNA-protein complexes were isolated and treated with 200 μl PK buffer (10 mM Tris-HCl pH 7.4; 100 mM NaCl; 1 mM EDTA; 0.2% SDS) and 10 μl proteinase K (Roche, 03115828001) for 1 hr minutes at 1100 rpm and 50°C. Solution was collected and RNA was isolated by phenol:chloroform extraction and ethanol precipitation over night at -20°C. RNA pellet was resuspended in 7 μl RNA/primer mix (5.5 μl water; 1 μl irCLIP_ddRT_## primer; 0.5 μl dNTP mix [10mM]) and incubated at 65°C for 5 min. RNA was retrotranscribed by addition of 3 μl of RT mix (2 μl 5x SSIV buffer (Invitrogen); 0.5 μl 0.1 M DTT; 0.25 μl Superscript IV reverse transcriptase [Invitrogen]; 0.25 ul of RNasin) and incubation for 5 min at 25°C, 5 min at 50°C and 5 min at 55°C. cDNA was gel-size selected by running samples on a precast 6% TBE-urea gel 180V for 40 min and isolating a region between 75 and 200 nt. Gel slices were submerged and crushed in TE buffer, incubated 1 h at 37°C 1100 rpm, flash frozen, and incubated again for 1 h at 37°C 1100 rpm. cDNA was isolated by phenol:chloroform extraction and ethanol precipitation over night at -20°C. cDNA was resuspended in 7 μl of water and then 8 ul of CircLigase II mix (5 μl water; 1.5 μl 10x CircLigase Buffer II; 0.75 μl 50 mM MnCl2; 0.75 μl Circligase II [Epicentre]) were added. Samples were incubated for 1 h at 60°C for circularization and cDNA was isolated by phenol:chloroform extraction and ethanol precipitation over night at -20°C. Pellets were resuspended in 10 μl of H20. Amplification was performed using 4 μl cDNA; 1 μl primer mix P5/P3 solexa, 10 μM each; 20 μl Phusion HF Master mix [NEB], 15 μl H20 and the following PCR programme: 98°C for 40 sec, [98°C for 20 sec, 65°C for 30 sec, 72°C for 45 sec]x18 cycles, 72°C for 3 min, 4°C. Libraries were loaded on a precast 6% TBE gel (Invitrogen) and run at 180V for 30 min. Gel was stained with Sybrgreen I (Invitrogen) and a region between 150-300 nt was excised. Gel slices were submerged in Crush-Soak Gel buffer (500 mM NaCl; 1 mM EDTA; 0.05% SDS) and incubated at 65ºC for 2 hours with thermomixer settings of 15 sec at 1000 rpm, 45 sec rest. cDNA was isolated by phenol:chloroform extraction and ethanol precipitation over night at -20°C and resuspended in 10 ul of water. Libraries were sequenced using Illumina HiSeq 2500 sequencers (50 bp single-end reads).