alias: E-MTAB-6941:Liver_RIP_Cnot3_3_p
title: Illumina HiSeq 4000 paired end sequencing; RIP-seq of livers in C57BL/6J mice with antibodies against Cnot3, Ago2, and BRF1
design: RIP-seq of livers in C57BL/6J mice with antibodies against Cnot3, Ago2, and BRF1
library_construction_protocol: Liver was isolated from C57BL/6J wild-type mice at 8 weeks of age Mice were maintained on a 12 hr light / dark cycle in a temperature-controlled (22ºC) barrier facility with free access to water and a normal diet (NCD, CA-1, CLEA Japan, Inc.).  Liver was solubilized in TNE buffer for 30 min at 4ºC. 150 mg of protein lysate was incubated with 180 µg of antibodies against Cnot3 (Takahashi et al., 2015), Ago2 (018-22021; Wako), and BRF1 (Cell Signaling) for 1 hr at 4ºC, and then incubated with 1.2 mL of Dynabeads (Invitrogen) for 2 hr at 4ºC. mRNAs in immune complexes were isolated using Isogen II (Nippongene).  100 ng of total RNA was used for RNA-seq library preparation with TruSeq Stranded mRNA Library Prep Kit for NeoPrep (NP-202-1001; Illumina), according to the manufacturer's protocol with minor modification and optimization as follows. Custom dual index adaptors were ligated at the 5' and 3'-end of library, and PCR was performed 11 cycles.
identifiers.namespace: Cell Signal Unit, Okinawa Institute of Science and Technology Graduate University
identifiers.id: E-MTAB-6941:Liver_RIP_Cnot3_3_p