, Tax (1A3, Covalab), and DDX17 (ProteinTech) in the presence of 30 ?l Dynabeads® Protein A/G (ThermoFisher), Cells were harvested in IP lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM EDTA, 1% NP-40, 10% glycerol)
, Nuclei were isolated by sonication using a Covaris S220 (2 min, Peak Power: 75; Duty Factor: 2; Cycles/burst: 200), pelleted by centrifugation at 1000×g for 5 min at 4°C, washed once with FL buffer (5 mM HEPES pH 8.0, 85 mM KCl, 0.5% NP-40) and resuspended in 1 ml shearing buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 2 mM EDTA, 0.1% SDS), Chromatin immunoprecipitation. A total of 10 7 cells were crosslinked with 1% formaldehyde for 10 min at room temperature. Crosslinking was quenched by addition of 0.125 M glycin
, Santa Cruz), anti-DDX17 (19910-1-AP, ProteinTech), or anti-V5 (AB3792, Millipore), and 30 ?l Dynabeads® Protein A/G (ThermoFisher) were added, Complexes were washed with 5 different buffers: Wash 1 (1% Trition, 0.1% NaDOC, 150 mM NaCl, 10 mM Tris-HCl pH 8), Wash 2 (1% NP-40, 1% NaDOC, 150 mM KCl, 10 mM Tris-HCl pH 8)
, Values were expressed relative to the signal obtained for the immunoprecipitation with control IgG. Primers used for ChIP experiments were designed for exon/intron junction (Supplementary Data 5). For TALE ChIP experiments, DDX17 and RelA enrichment were normalized to the signal observed with V5 antibody corresponding to TALE recruitment. The TALE-GFP condition was used as control and set to 1, NaDOC, 500 mM NaCl, 10 mM Tris-HCl pH 8), Wash 4 (0.5% NP-40, 0.5% NaDOC, 250 mM LiCl, 20 mM Tris-HCl pH 8, 1 mM EDTA), and Wash 5 (0.1% NP-40, 150 mM NaCl, vol.20
Quantitative PCR was then performed using 5 ng of cDNAs with SYBR® Premix Ex Taq TM II (Tli RNaseH Plus) on LightCycler 480 II. Relative levels of the target sequence were normalized to the 18 S or GAPDH gene expression (?Ct), and controls were set to 1(??Ct). The inclusion rate of alternative exons was calculated as 2 ???Ct, RNA extraction, PCR, and real-time quantitative PCR. Total RNAs were extracted using TRIzol (Invitrogen) ,
RNA-seq analyses were performed with poly-A transcripts extracted from 293T-LTR-GFP cells transfected with pSG5M-Tax or pSG5M empty vectors and knocked down or not for DDX5-17. RNA-seq libraries were generated at Aros Applied Biotechnology (Aarhus, Denmark) using Stranded mRNA Sample Prep kit (Illumina) and sequenced using illumina HiSeq 2500 technology. Each sample had in average 6 × 10 7 of paired-end pairs of reads. RNA-seq data were analyzed using FaRLine, a computational program dedicated to analyzing alternative splicing with FasterDB database 23,54 . The gene expression level in each sample was calculated with HTSeq-count ,
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