RIP-seq of livers in C57BL/6J mice with antibodies against Cnot3, Ago2, and BRF1

Shortening of polyadenosine (poly(A)) tails (deadenylation) is the initial step in the decay of most mRNAs. The CCR4-NOT complex is the major deadenylase in mammals. Interaction of Cnot1 with RNA binding proteins, including the miRNA-induced silencing complex (miRISC), which includes Argonaute (Ago) and GW182 as core proteins, and the TTP family of AU-rich element (ARE)-binding proteins (TTP and BRF1/2), recruits the CCR4-NOT complex to the 3'-untranslated regions (3'-UTR) of mRNAs, leading to their degradation. This experiment aimed to determine the binding affinity of the CCR4-NOT complex, Ago2, and BRF1 with their target mRNAs in liver. Liver isolated from C57BL/6J wild-type mice at 8 weeks of age was solubilized in TNE buffer for 30 min at 4ºC. Lysate was incubated with antibodies against Cnot3, Ago2, and BRF1 for 1 hr at 4ºC, and then incubated with Dynabeads (Invitrogen) for 2 hr at 4ºC. mRNAs in immune complexes were isolated using Isogen II. 100 ng of total RNA was used for RNA-seq library preparation with TruSeq Stranded mRNA Library Prep Kit for NeoPrep. 150 base-pair pair-end read RNA-seq was performed with Hiseq 3000/4000 PE Cluster Kit and Hiseq 3000/4000 SBS Kit on Hiseq4000.