title: GSM1632837: Suz12 Ezh2 d/d 3; Mus musculus; RIP-Seq
library_construction_protocol: Individual-nucleotide resolution UV crosslinking and immunoprecipitation protocols were based in protocols available at Huppertz et al. Cells were irradiated with 0.15 J/cm2 of UV light in a Stratalinker 2400 (Stratagene) with bulbs emitting at 254nm, and 200 x10^6 cells per IP were lysed in 1ml of Lysis buffer (50 mM Tris–HCl, pH 7.4; 100 mM NaCl; 1% Igepal CA-630; 0.1% SDS; 0.5% sodium deoxycholate; plus protease inhibitors). 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. IP was set up using 10 μl of α-SUZ12 (Cell Signaling #3737)  as described in iCLIP protocol. Beads were washed 3x 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 and Urea 1M), 2x with 900 μl wash buffer (20 mM Tris-HCl, pH 7.4; 10 mM MgCl2; 0.2% Tween-20) resuspended in 20 μl PNK mix (15 μl water; 4 μl 5x PNK pH 6.5 buffer [350mM Tris-HCl, pH 6.5; 50m MgCl2 25mM dithiothreitol]; 0.5 μl PNK enzyme; 0.5 μl RNasin [Promega]) and incubated 30 min at 37°C 1100 rpm. Beads were washed 3x with 900 μl high-salt buffer, 2x with 900 μl wash buffer, resuspended in beads in 20 μl ligation mix (9 μl water; 4 μl 4x ligation buffer [200 mM Tris-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. Beads were washed 3x with high-salt buffer, 2x wash buffer and 20% of the beads were incubated with 8 μl of hot PNK mix (0.4 μl PNK [NEB]; 0.8 μl 32P-γ-ATP; 0.8 μl 10x PNK buffer [NEB]; 6 μl water), radiolabelled and non-radiolabelled beads were pooled again and resuspended in 20 μl in 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 nitrocellulose membrane (Hybond, GE Healthcare) washed 2x with PBS1x and exposed to a Fuji film. Using the auto-radiograph 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 PK-urea 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 7.25 μl RNA/primer mix (6.25 μl water; 0.5 μl Rclip primer [0.5 uM]; 0.5 μl dNTP mix [10mM]) and transferred in a 0.2 ml tube and incubated at 70°C. Retrotranscription was performed by addition of 2 μl 5x RT buffer; 0.5 μl 0.1M DTT; 0.25 μl Superscript III reverse transcriptase [Invitrogen] and incubation 20 min at 42°C, 40 min at 50°C and 5 min at 80°C. cDNA was fractionated running samples on a precast 6% TBE-urea gel 180V 40 min and isolating bands at 250-180 nt (top), 120-180 nt (high), 85-120 nt (medium) and 70-85 nt (low). Gel slices were submerged and crushed in TE buffer, incubated 1h at 37°C 1100 rpm, flash frozen, and a second incubation was performed 1h at 37°C 1100 rpm. cDNA was isolated by phenol; chloroform extraction and ethanol precipitation over night at -20°C. cDNA was resuspended in 8 μl ligation mix (6.5 μl water; 0.8 μl 10x CircLigase Buffer II; 0.4 μl 50 mM MnCl2; 0.3 μl Circligase II [Epicentre]) transferred into a 0.2 tube and incubated for 1 h at 60°C for circularization. Samples were incubated 30 μl oligo annealing mix (26 μl water; 3 μl FastDigest Buffer [Fermentas]; 1 μl cut_oligo [10 μM]), oligo was annealed by temperature gradient and circular cDNA was re-linearized incubating samples with 2 μl BamHI (Fast Fermentas). cDNA was isolated by phenol; chloroform extraction and ethanol precipitation over night at -20°C and pellet were resuspended in 20 μl of H20. Amplification was performed using 10 μl cDNA; 1 μl primer mix P5/P3 solexa, 10 μM each; 20 μl Accuprime Supermix 1 enzyme [Invitrogen], 9 μl H20 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 8 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. Library concentration was determined using the KAPA Universal Library Quantification kit, according to the manufacturer's instructions and library concentration was corrected by multiplication by 0.38 to improve cluster density. Libraries were sequenced using Illumina HiSeq 2500 sequencers (50 bp single-end reads).
attributes.tag: GEO Accession
attributes.value: GSM1632837