title: GSM3466977: CHMP3; Mus musculus; miRNA-Seq
library_construction_protocol: After 72 h macrophage culture, debris and dead cells in the medium were removed by centrifugation at 1000 × g for 10 min and then filtered through a 0.2-mm filter. The medium was then subjected to ultracentrifugation at 100,000 × g for 4–6h at 4 °C. After washing with PBS (100,000 × g for 20 min), the Exo-containing pellet was resuspended in PBS.Total RNA was extracted from exosome pellet using the TRIzol reagent (Invitrogen) following the manufacturer's protocol.RNA concentration was quantitated using the NanoDrop ND-100 Spectrophotometer (NanoDrop Technologies, Wilmington, DE). Total RNA was isolated from exosomes using the Trizol (invitrogen) according to the manufacturer's protocol. RNA purity was assessed using the ND-1000 Nanodrop. Each RNA sample had an A260:A280 ratio above 1.8 and A260:A230 ratio above 2.0. RNA integrity was evaluated using the Agilent 2200 TapeStation (Agilent Technologies, USA) and each sample had the RIN e above 7.0. Briefly, RNAs were ligated with 3'RNA adapter, and followed by 5'adapter ligation. Subsequently, the adapter-ligated RNAs were subjected to RT-PCR and amplified with a low-cycle. Then the PCR products were size selected by PAGE gel according to instructions of NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (Illumina, USA). The purified library products were evaluated using the Agilent 2200 TapeStation and diluted to 10 pM for cluster generation in situ on the HiSeq2500 single-end flow cell followed by sequencing (1×50 bp) on HiSeq 2500.
attributes.tag: GEO Accession
attributes.value: GSM3466977