title: Aog2-binding small RNAs of U937 total RNA after A.actinomycetomcomitans OMV infection
design: For Ago2 RNA immunoprecipitation sequencing (RIP-Seq), nine dishes of PMA-treated U937 cells were incubated with A. actinomycetemcomitans OMVs. After 24 h, all culture medium was removed, each plate was washed with 10 ml of PBS and the cells were collected and lysed using lysis buffer (50 mM HEPES, 150 mM KCl, 2 mM EDTA, 1 mM NaF, 0.5% NP40, 0.5 mM DTT). Lysates were incubated with Dynabeads Protein G (Life Technologies) conjugated with an Ago2-specific monoclonal antibody (ab57113, Abcam). The beads were repeatedly washed with PBS and then TRIzol was added to extract bound RNA, which was subjected to small RNA deep-sequencing. The quantity and quality of the total RNA were evaluated using RNA electropherograms (Agilent 2100 Bioanalyzer, Prokaryote Total RNA Pico Kit) and by assessing the RNA integrity number (RIN). The resulting RNA samples were processed for the sequencing libraries using the Illumina TruSeq small RNA sample preparation kit (Illumina, San Diego, CA, USA) according to the manufacturers protocols. The purified small-sized cDNA library, amplified by 11 cycles of PCR, was used for cluster generation on Illuminas (San Diego, CA, USA) Cluster station following the TruSeq Small RNA Sample Preparation Guide (Catalog #RS-200-9002DOC), and then sequenced with 50 cycles in single-read mode on the Illumina (San Diego, CA, USA) HiSeq 2000 system following the manufacturers instructions.
identifiers.namespace: SUB5381844
identifiers.id: U937 sRNA from RIP