Total RNAs were isolated from cells grown in 15 cm culture dishes using miRNeasy Kit (Qiagen) according to manufacturer's instructions. Small RNAs, including miRNAs, were isolated using the manufacturer's enrichment protocol. The larger RNA fraction (>200 nt) was enriched for mRNAs using NEBNext Poly(A)mRNA Magnetic Isolation Module (New England Biolabs). For PAR-CLIP, crosslinked cells were resuspended in NP40 lysis buffer and incubated on ice for 10 minutes. The lysates were cleared by centrifugation and syringe filter. RNAs in the cleared lysate were partially degraded by treatment with RNAse T1 for 15 minutes at 22°C followed by 5 minutes on ice. Immunoprecipitation of target Argonaute (AGO) protein was accomplished using antibody-bound Dynabeads Protein G magnetic particles (Thermo Fisher) for 2 hours in lysis buffer (lower salt concentration than IP buffer) at 4°C. The complete RNA isolation protocol was completed as described by Hafner et. al, 2010 (PMCID: PMC4180672). mRNAs and miRNA libraries were constructed using NEBnext Multiplex Small RNA and mRNA Library Prep Sets for Illumina (New England Biolabs). PAR-CLIP cDNA libraries were constructed from recovered RNA by sequentially ligating a preadenylated 3'-adapter using T4 Rnl2(1-249)K227Q ligase (New England Biolabs) and an RNA 5'-adapter using T4 RNA ligase. Adapters are designed to be compatible with Illumina Small RNA library and sequencing protocols. After PCR amplification the libraries were size fractionated by agarose gel electrophoresis.
GEO Accession
GSM2609181