GSM5526107: CLEAR-CLIP_LN229_AGO2_rep2; Homo sapiens; RIP-Seq
The cells were lysed, treated with DNase (Promega), followed by the partial RNA fragmentation using low concentrations of the RNase I (0.002 U/ml, 5 min), and treatment with the RNase inhibitor (RNAsin Plus at 0.5 U/μl) to quench RNase activity. The miRNA–RNA complexes were immunopurified using the anti-Ago-2 antibody (Millipore, #03-110) immobilized on immunoglobulin G-coated magnetic beads (Life Technologies).  The beads were washed, treated with T4 Polynucleotide Kinase (PNK, with no 3'- phosphatase activity)  (NEB, M0236S), and miRNA-RNA chimeras were produced with T4 RNA Ligase I (NEB, M0204S). The beads were further washed and subjected to alkaline phosphatase treatment (Roche, #10713023001) to remove 3' phosphate. The 3' linker (5′-rAppGTGTCAGTCACTTCCAGCGG-3', Dharmacon) was added using T4 RNA Ligase 2 (NEB, M0242S), followed by the PNK (NEB, M0201S) treatment. The samples were resolved on 8% acrylamide gel (ThermoFisher Scientific), transferred to nitrocellulose membrane, followed by the purification of RNA-Ago complex, RNA isolation. The libraries have been prepared from the low amount of RNA input using Superscript Reserve transcriptase (ThermoFisher) with first PCR amplification performed with DP3 (5'-CCGCTGGAAGTGACTGACAC-3') and DP5 (5'-AGGGAGGACGATGCGG-3') primers, and additional amplification with DSFP3 (5'-CAAGCAGAAGACGGCATACGACCGCTGGAAGTGACTGACAC-3', DSFP5 (5'-AATGATACGGCGACCACCGACTATGGATACTTAGTCAGGGAGGACGATGCGG-3') at maximum of 16 PCR cycles. The miR-10b chimeras have been pre-amplified with a miR-10b primer TACCCTGTAGAACCGAATTTGTG and either DP3 or DP5 primers. The products were separated from primers and primer dimers using electrophoresis on a 1% agarose gel, recovered using a QIAquick gel extraction kit (Qiagen). CLEAR-CLIP: Covalent Ligation of Endogenous Argonaute bound RNA -Crosslinking and immunoprecipitation