title: GSM3526591: library enriched for mRNA (chimeric) fragments of RISC complex; Homo sapiens; OTHER
library_construction_protocol: HITS-CLIP was performed according to the method of Chi et al. (PMID: 19536157) with slight modification. Briefly, human CD4 + T cells were subjected to UV-crosslinking three times at 400 mJ/cm². Cells were then lysed and treated sequentially with DNase and RNase T1. The Argonaute complex was then immunoprecipitated 2-4 hours at 4°C using the monoclonal Argonaute antibody 2A8. After on-bead ligation of a 3' linker, Ago2-RNA complexes were isolated by SDS gel electrophoresis and RNA was extracted. 5' RNA linkers were then ligated to the RNA sample. RNA molecules with 5' and 3' linkers were amplified by RT/PCR, and the resulting products were separated on agarose gel. Two respective populations of RNA (50-60bp for small RNAs, 80-180bp for mRNAs) were extracted. A second round of PCR using Solexa Fusion primers was performed and products were purified by gel extraction, analyzed on an Agilent Bioanalyzer, and  subjected to sequencing on an Illumina Hi-Seq 2000. The readouts were preprocessed and mapped to the human genome (UCSC hg19 assembly) and potential miRNA-mRNA pairs were predicted using the miRanda program (v3.3a). Ago2-bound mRNA and miRNA sequencing libraries were derived based on the method of Chi SW, et al. [PMID: 19536157]. HITS-CLIP
attributes.tag: GEO Accession
attributes.value: GSM3526591