GSM3040153: pre-IP_7; Homo sapiens; RIP-Seq
Cell lysates were prepared using Hypotonic Gentle Lysis Buffer (10 mM Tris (pH 7.4), 10 mM NaCl, 10 mM EDTA, 0.5% w/w Triton X-100) and Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific). RNA was extracted and purified using TRIzol reagent (Thermo Fisher Scientific). HEK293T cells (6 x 108 in a total of eight 150-mm dishes) were treated with okadaic acid (200 nM) for 3 hr. After IP using anti-p-UPF1 (EMD Millipore) and, in parallel as a negative control, rabbit IgG (rIgG; Sigma), RNA was purified using TRIzol Reagent (Thermo Fisher Scientific), incubated following manufacturer's instruction first with DNase I (Promega) and, subsequently, with Ribozero Gold rRNA Removal Kit (Illumina). Samples were then incubated with recombinant shrimp alkaline phosphatase (rSAP; New England Biolabs) to remove 5'-phosphates from any physiologically uncapped NMD targets; 3'-ends were ligated using T4 RNA ligase 2 (T4 RNA Ligase 2, truncated KQ; New England Biolabs) to a 33-nucleotide DNA adapter having App appended at its 5'-end and dideoxy-C (ddC; Integrated DNA Technologies) appended at its 3'-end; and 5'-ends were then phosphorylated using T4 polynucleotide kinase (T4 PNK; New England Biolabs) and ligated using T4 RNA ligase (Ambion T4 RNA Ligase; Thermo Fisher Scientific) to a 33-nucleotide RNA adapter having a hydroxyl moiety at both its 5'- and 3'-ends (Integrated DNA Technologies). cDNA was synthesized using SuperScript III reverse transcriptase (Invitrogen) and a DNA primer that is antisense to the 3'-end DNA adapter, and subsequently PCR-amplified (20 cycles) using primers complementary to the extreme ends of each adapter and Q5 High-Fidelity DNA Polymerase (New England Biolabs). Products with inserts of >20 base-pairs were size-selected from 6% polyacrylamide to eliminate those that could not be uniquely aligned to the genome, including products without inserts, PCR-amplified using index PCR primers, and submitted for deep sequencing (Illumina HiSeq2500v4 system).