GSM1464150: pUPF1_2; Homo sapiens; RIP-Seq
Cellular RNAs bound by p-UPF1 were immunoprecipitated using anti-p-UPF1(S1116) (Millipore, anti-phospho-Upf1(Ser1127)) and Dynabeads protein A magnetic beads (Life Technologies). The RNA in bead-bound RNA p-UPF1(S1116) complexes was digested to primarily  100 nts by incubation for 30 min at 4°C with RNase I (1U/l; Life Technologies); for RNA size estimations after RNase I digestion, ~200 ng of RNA were radio-labeled using  32P-ATP (Perkin Elmer) and T4 polynucleotide kinase (New England Biolabs) and subsequently visualized using a Typhoon 9410 Variable Mode Imager (GE Healthcare) after electrophoresis in 6M urea 15% polyacrylamide. After extensive washing, bound complexes were eluted using IP elution buffer (Kurosaki and Maquat 2013), and RNA fragments were purified using TRIzol Reagent and then separated in 6M urea 15% polyacrylamide in parallel with a DynaMarker Prestain Marker for Small RNA (BioDynamics Laboratory). Small-range RNAs (~25-40 nts) were excised from 6M urea 15% polyacrylamide and agitated overnight at 25°C in RNA extraction buffer (20 mM Tris, 300mM sodium acetate, 2 mM EDTA, 0.2 % v/v SDS). RNAs were eluted from 6M urea 15% polyacrylamide gel using a Coaster Spin-X column (Corning) and further purified using TRIzol Reagent followed by ethanol precipitation. In parallel, control IPs using rabbit IgG were performed. Additional control samples were prepared without IP. Purified 25-40-nt RNA fragments were treated with recombinant shrimp alkaline phosphatase (New England Biolabs) to remove 3'-phosphates and subsequently phosphorylated at 5'-hydroxyl groups using T4 polynucleotide kinase (New England Biolabs). Phosphorylated RNA fragments were purified with RNeasy Mini Columns (Qiagen). A 3'-adenylated adapter was ligated to the phosphorylated RNA fragments using truncated T4 RNA ligase (New England Biolabs). An RT primer was annealed to the adapted RNAs to prevent adapter self-ligation, followed by 5' RNA adapter ligation using T4 RNA ligase (New England Biolabs). After RT of adapter-ligated RNAs, cDNAs were amplified using 15 PCR cycles. Amplified cDNAs were purified in 8% polyacrylamide, and the quality and quantity of cDNAs were assessed using an Agilent Bioanalyzer and qPCR. cDNAs were then sequenced using the Illumina HiSeq 2500 platform.