title: A21_IP_rep1
organism: Mus musculus
description: Protocols: NIH3T3 cells were transfected with CMV-tdTomato (fluorescent transfection marker) plasmids and LNA antimiRs (power inhibitors, Exiqon) for the respective microRNAs using Lipofectamine 2000 and cultured for 48 h in DMEM with 5% FCS, without antibiotics. Two days later cells were trypsinized, washed with PBS and fluorescent cells were sorted using a Bio-rad S3 sorter. Sorted cells were spinned down, the supernatant was removed and the cells were frozen at -80 °C. Ago2-RIP was performed in parallel for the six groups using an anti-mouse-Ago2 antibody (clone 2D4, WAKO) for 3 h at 4°C. RNA from the input and IP fractions was isolated using peqGOLD RNApure reagent (VWR). RNA-Seq libraries were prepared in parallel from both the input and IP fractions of the Ago2-RIPs. First, a DNase digestion with consecutive DNase inactivation was performed to remove plasmid and genomic DNA. For the input fraction the RNA integrity was then confirmed using a Bioanalyzer (Agilent, RIN was >8 for all samples) and a ribosome depletion was performed using a RiboMinus Eukaryote Kit v2 (ThermoFisher) following manufacturer's protocol. For both input and IP fractions the RNA was then fragmented in 1x magnesium RNA fragmentation buffer (NEB) for 3.5 min at 94 °C and extracted using phenol-chloroform. First strand cDNA synthesis was then performed using Superscript III (ThermoFisher), followed by second strand synthesis with DNA polymerase I (final concentration 1 U/µl) in the presence of 0.04 U/µl RNase H (ThermoFisher). The resulting double-stranded DNA was then purified using AMPure XP paramagnetic beads (Beckman Coulter). End repair and further preparation steps were performed using NEBNext Ultra II DNA library prep kit for Illumina (NEB) as per manufacturer's protocol. Libraries were multiplexed and all libraries pertaining to one experiment (6x input, 6x IP) were sequenced together.