GSM5416652: WT J2 RIP 2; Homo sapiens; RIP-Seq
Total RNA was harvested from WT Ad5 or E4-infected cells 48 hpi by TRIzol extraction. RNA was diluted to 500 ng/L in NET2 Buffer (50 mM Tris HCl pH 7.6, 150 mM NaCl, 3 mM MgCl2) and divided into immunoprecipitated fractions (IP, 50 g RNA) or 1% input RNA (0.5 g RNA). IP samples were treated with 2 Units of diluted single-strand specific RNase I (Thermo AM2294, 100 U/L) and 2 L TURBO DNase (Thermo AM2238) at 37 °C for ten minutes before the addition of 10 L SuperAse RNase inhibitor (Thermo AM2696) and placement on ice. 5 g J2 anti-dsRNA antibody (Scicons) was added to IP fraction to a final volume of 200 L and incubated overnight at 4 °C with end over end rotation. Protein G Dynabeads (Thermo 10004D) were washed twice with NET2 before being blocked with 1 mg/mL BSA and 10 g/mL linear acrylamide (Thermo AM9520) for thirty minutes. Blocked beads were added to J2/RNA mixture and allowed to continue rotating for an additional two hours. RNA-bound beads were subsequently washed using ice-cold NET2 buffer thrice for five minutes each before RNA was extracted using TRIzol LS (Thermo 10296010). Aqueous-phase TRIzol was subsequently extracted using RNA Clean and Concentrator (Zymo Research R1015) and eluted into 11 L elution buffer. Input or dsRNA-enriched RNA from above was converted into Illumina sequencing libraries using the NEBNext Ultra II Directional RNA library kit (NEB E7760S) following manufacturers instructions with the following modifications. Ribosomal RNA was depleted using NEBNext rRNA depletion kit v2 (NEB E7405S). Input RNA was chemically fractionated for 15 minutes, while dsRNA IP RNA was fractionated for 7 minutes as directed for partially degraded RNA due to prior RNase I digestion. IDT xGen UDI-UMI Adapters 1-8 (Integrated DNA Technologies 10006913) were ligated to resulting cDNA after second-strand synthesis and end-prepping. All library purification steps used NEBNext RNA Sample Purification Beads (Beckman Coulter Agencourt RNAClean XP) or SPRISelect (Beckman Coulter B23317). After initial library quantification via qPCR, final libraries were amplified using xGen Library Amplification Primer Mix (Integrated DNA Technologies 1076775) and NEBNext Ultra II Q5 master mix for 4 cycles (input libraries) or 10 cycles (IP libraries). Libraries were quantified using Thermo Qubit fluorometric quantification, Agilent Bioanalyzer DNA Chip, and qPCR quantification relative to a standard curve. Eight total libraries were pooled in equimolar ratios and loaded onto an Illumina NextSeq 500 High Output flow cell (Illumina FC-404-2005) and ran for 150 cycles in single end mode.