GSM3408503: Suz12 Input; Mus musculus; RIP-Seq
Input Individual-nucleotide resolution UV crosslinking and immunoprecipitation protocols were based in  modification of protocols available at Huppertz et al. Cells were irradiated with 0.2 J/cm2  of UV light in a Strataliker 2400 (Stratagene) with bulbs emitting at 254nm, and 25 x106 cells  per sample were lysed in 50μL of Lysis buffer (see iCLIP section). Lysates were syringed with a 27G needle and incubated in a Thermomixer at 37°C and 1100 rpm for three minutes after addition of 200U/mL of DNase Turbo and of 20 U/mL of RNase I. Sample was mixed with Nupage loading buffer with reducing agent 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 nitrocellμlose membrane (Hybond, GE Healthcare) washed 2x with PBS1x and exposed to a Fuji film. Using the auto-radiograph previously used for iCLIP as a mask RNA-protein complexes  attached to the membrane were isolated and treated with 200 μl PK buffer (100 mM Tris-HCl pH 7.4; 50 mM NaCl; 10 mM EDTA) and 10 μl proteinase K (Roche, 03115828001)  for 20 minutes at 1,100 rpm and 37°C. 200 μl of PKurea buffer (100 mM Tris-HCl pH 7.4; 50 mM NaCl; 10 mM EDTA; 7 M urea) was added and a second incubation was performed for 20 minutes at 37°C and 1100 rpm. Solution was collected and RNA was isolated by phenol; chloroform extraction and ethanol precipitation over night at -20°C. RNA pellet was resuspended in 20 μl PNK mix (15 μl water; 4 μl 5x PNK pH 6.5 buffer [350mMTris-HCl, pH 6.5; 50mMgCl2 25mMdithiothreitol]; 0.5 μl PNK enzyme; 0.5 μl RNasin [Promega]) and incubated 30 min at 37°C 1100 rpm. Solution was collected and RNA was isolated by phenol; chloroform extraction and ethanol precipitation over night at -20°C. Pellet was resuspended in 20 μl ligation mix (9 μl water; 4 μl 4x ligation buffer [200 mMTris-HCl; 40m MM gCl2; 40 mM dithiothreitol]; 1 μl RNA ligase [NEB]; 0.5 μl RNasin [Promega]; 1.5 μl pre-adenylated linker L3 [20 μM]; 4 μl PEG400 [81170, Sigma]), ligation was performed over night at 16°C and 1100 rpm. L3-RNA was isolated by phenol; chloroform extraction and ethanol precipitation over night at -20°C and pellet were resuspended in 13 μl of H20, reverse transcription was performed using specific oligo and Superscript III (Thermo) according instructions. cDNA was isolated by phenol; chloroform extraction and ethanol precipitation over night at -20°C and pellet were resuspended in 10 μl of H20+ TBE-Urea buffer. cDNA was fractionated by running samples on a precast 6% TBE-urea gel at 180 V for 40 min and cDNA bands running between 250-180 nt (top), 120-180 nt (high), 85-120 nt (medium) and 70-85 nt (low) isolated. cDNA was isolated Tris-EDTA gel resuspension and separation by phenol; chloroform extraction and ethanol precipitation over night at -20°C. cDNA was circularised using Circligase II (CamBio) and digested after hybridization with annealing oligo with BamHI. cDNA was isolated by phenol; chloroform extraction and ethanol precipitation over night at -20°C and pellet were resuspended and amplification was performed using 10 μl cDNA; 1 μl primer mix P5/P3 solexa, 10 μM each; 20 μl Accuprime Supermix 1 enzyme [Invitrogen], 9H20  and the following PCR programme: 94°C for 2 min, [94°C for 15 sec, 65°C for 30 sec, 68°C for 30 sec]n cycles, 68°C for 3 min, 4°C, with a number of cycles optimized previously. Library integrity was analysed using 8 μl PCR product with 2 μl of 5x TBE loading buffer and loading on a precast 6% TBE gel (Invitrogen). Gel was stained with Sybrgreen I (Invitrogen) and analyse with a gel imager. Concentration was determined using KAPPA kit according manufacturer instructions.