title: GSM4466626: S5: Clean Air 8-oxoG-Immunoprecipitated RNA; Homo sapiens; RNA-Seq
library_construction_protocol: TRIzol RNA extraction was conducted following TRIzol's manufacturer instructions with freshly prepared ethanol (200 Proof, OmniPur, EMD Millipore, Burlington, MA), isopropanol (molecular biology grade, IBI Scientific, Dubuque, IA) and nuclease-free water (Ambion, Austin, TX) purged of oxygen with ultra-high purity N2. The purified RNA was incubated with DNAseI (New England Biolabs, Ipswich, MA) following the manufacturer's protocol. RNA was then re-extracted with 200 µl of 25:24:1 mixture of phenol/chloroform/isoamyl alcohol (Fisher BioReagents, Hampton, NH) followed by a chloroform extraction and an isopropanol precipitation as described above. After DNase I treatment of RNA, ribosomal RNA (rRNA) was depleted using Ribo-Zero Gold rRNA Removal Kit (Illumina, San Diego, CA) as described by the manufacturer's protocol to produce the formaldehyde air and clean air input RNA samples (FAInput and CAInput).  Immunoprecipitations of 8-oxoG-containing RNA transcripts were performed in biological duplicates for CA and FA conditions. All buffers were prepared fresh from concentrated stocks on the day of pulldown experiments and purged of O2 as described above. A portion of the input RNA was incubated with 12.5 µg of 8-oxo-7,8-dihydrodeoxyguanosine (8-oxo-dG) monoclonal antibody (0.5 mg/ml, Clone 2E2, Trevigen, Gaithersburg, MD) in IP buffer (10 mM Tris pH 7.4, 150 mM NaCl, 0.1% IGEPAL, and 200 units/ml SUPERaseIn RNA inhibitor [Invitrogen, Carlsbad, CA]) in a 1 ml reaction volume for 2h at 4°C with rotation.  SureBeads Protein A magnetic beads (Biorad, Hercules, CA) were washed according to manufacturer's protocol and blocked in IP buffer supplemented with 0.5 mg/ml bovine serum albumen (BSA) for two hours at room temperature. After washing beads twice in IP buffer, the beads were resuspended in IP buffer, mixed with the RNA-antibody reaction and incubated for 2h at 4°C with rotation. Next, the beads were washed three more times in IP buffer before two rounds of competitive elution were performed with free 8-oxo-dG nucleosides (Cayman Chemical, Ann Arbor, MI). Each elution consisted of incubation of the beads with 108 µg of 8-oxo-dG in IP buffer for 1h at 4°C with rotation. The elution volume was then cleaned up using the RNA Clean and Concentrator-5 kit (Zymo Research, Irvine, CA). RNA libraries were prepared for sequencing using standard Illumina protocols library strategy: 8-oxoG-Seq
attributes.tag: GEO Accession
attributes.value: GSM4466626