title: GSM1873380: CAP_IgG_KO; Mus musculus; RIP-Seq
library_construction_protocol: Total RNA was extracted by using TRIzol reagent (Invitrogen). For immunoprecipitation of mRNA for CAPIP-Seq experiments, 30ug of total RNA were fragmented by alkaline hydrolysis in ~200nt fragments and incubated with 5ug of mouse anti-CAP antibody (Anti-m3G-cap, m7g-cap, Clone H20, Millipore MABE419) (or IgG) overnight at 4°C in 0,5ml of IP Buffer (10mM Tris-HCl pH 7.5; 150mM NaCl; 0,1% Triton X-100) supplemented with 50U/ml RNaseOUT (Invitrogen), 50U/ml SuperaseIN (Invitrogen), and 50U/ml RNase Inhibitor (Ambion).  25ul of Dynabeads Protein G (Invitrogen) were saturated overnight at 4°C in IP Buffer supplemented with 150ug of Sonicated Salmon Sperm DNA (Qiagen). Following incubation, beads were washed two times in IP Buffer and incubated with the preformed RNA-antibody complexes at 4°C. After 3hrs, beads were washed 4 times with IP Buffer. Specific elution of recovered fragments were obtained by incubation of beads with 100μl Elution Buffer [5mM Tris pH7.5; 1mM EDTA; 0.05% SDS; 0.3mg/ml Proteinase K] for 1h 30’ at 50°C. Fragments were then purified by addition of 1ml TRIzol reagent (Invitrogen), and subjected to random-primed reverse transcription using the SuperScript III Reverse Trancriptase (Invitrogen) at 50°C for 1h. Resulting cDNAs were then used as input for the TruSeq RNA Sample Prep kit (Illumina), starting from the “Second Strand Synthesis” step, to produce the sequencing library, following manufacturer instruction.
attributes.tag: GEO Accession
attributes.value: GSM1873380