GSM2128339: AGO2 PARCLIP L1; Homo sapiens; OTHER
After UV -crosslinking of A673 cells protein G Dynabeads (Invitrogen) were coupled to Ago2 antibody,while cells were lysed and RNA partially digested Then AGO2 IP took place followed by RNA 3´ends dephosphorylation, linker preparation (32P-γ-ATP) and RNA 5´ends labelling, SDS-PAGE in a4-12% NuPAGE Bis-Tris gel (Invitrogen). Lysates were clarified from sonicated nuclei and histone-DNA complexes were isolated with antibody. Protein-RNA complexes were transferred from the gel to a nitrocellulose membrane that was exposed till obtaining a clear image of the band corresponding to Ago2 molecular weight (97kDa). Band was excised and purified and RNA precipitated and resuspended in RNase-free H2O to proceed with 5´RNA linker ligation. Finally RT was done, followed by PCR with Solexa flow-cell adaptors (2` 95°C, 28 cycles of 20´´ 95°C, 30´´ 58°C, 20´´ 68°C, 5´ 68°C, ∞ 4C) and its product was loaded in a 4% low-melt agarose gel then extracted from the gel and eluted using the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany. DNA quantity and quality were measured using the Picogreen Assay (Invitrogen) for Nanodrop. cDNAs were then ready for sequencing Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Part# 0801-0303). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols. PARCLIP-Seq
GEO Accession
GSM2128339