Instrument: Illumina HiSeq 2500
Strategy: RIP-Seq
Source: TRANSCRIPTOMIC
Selection: other
Layout: SINGLE
Construction protocol: Individual-nucleotide resolution UV crosslinking and immunoprecipitation protocols were based in 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 6-10x107 cells per IP were lysed in 1mL of Lysis buffer. Lysates were syringed with a 27G needle and sonicated for 3x 10s pulses with a Diagenode Picoruptor. Lysates were 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. IPs were set up using 5ug of antibody as previously described in the 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 1M Urea), 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; 50mM 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 MgCl2; 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 nitrocellμlose membrane (Hybond, GE Healthcare) washed 2x with 1x PBS 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 overnight at -20°C. RNA pellet was resuspended in 7.25 μl RNA/primer mix (6.25 μl water; 0.5 μll Rclip primer [0.5 uM]; 0.5 μl dNTP mix [10mM]) and transferred in a 0.2 ml tube and incubated at 70°C. Reverse transcription 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 for 20 min at 42°C, 40 min at 50°C and 5 min at 80°C. cDNA was fractionated by running samples on a precast 6% TBE-urea gel at 180 V 40 min and isolating bands at 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 overnight 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 0.2ml tube and incubated for 1 h at 60°C for circularization. Samples were incubated in 30 μl oligo annealing mix (26 μl water; 3 μl FastDigest Buffer [Fermentas]; 1 μl cut_oligo [10 μM]), the oligo was annealed in a decreasing temperature gradient and circular cDNA re-linearized by incubating samples with 2 μl BamHI (Fast Fermentas). cDNA was isolated by phenol:chloroform extraction and ethanol precipitation overnight at -20°C and the pellet 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 a KAPA kit according to the manufacturer's instructions.