AGO2-CLASH: HM2
Cross-linking, ligation, and sequencing of hybrids (CLASH) was used to identify miRNA-target RNA interaction sites. In brief, a 10 cm dish of confluent cells was irradiated with UV-C (300 mJ/cm) to covalently bond RNA-binding proteins to bound nucleic acids. During the subsequent cell lysis, the lysate was treated with Turbo DNase (Invitrogen, AM2238) to remove DNA and RNAse 1 (Thermo Scientific, EN0601) to partially digest the RNA to generate 25-200 nt fragments. Immunoprecipitation of AGO2 proteins was performed using Dynabeads Protein G (Invitrogen, 10003D) conjugated with a mouse AGO2 antibody (WAKO, 014-22023) for 4 hours at 4C. After phosphorylation with T4 PNK (New England Biolabs, M0201), T4 RNA ligase 1 (New England Biolabs, M0204) was used for intermolecular ligation, followed by dephosphorylation and 3' adapter ligation using T4 RNA ligase 2, truncated, K227Q (New England Biolabs, M0351). T4 PNK labeling with 32P of RNA allowed visualization of the AGO2-RNA complexes by SDS-PAGE after transfer to a nitrocellulose membrane. The membrane sections containing protein-RNA complexes were cut and treated with proteinase K (Invitrogen, AM2542) to yield protein-free RNA. The 5' ends were then phosphorylated to prepare the RNA for sequencing library preparation. The final AGO2-CLASH library was sequenced on the Illumina NextSeq 1000 platform using paired-end sequencing.