title: GSM2942584: 006T_miRNAseq_SIV; Macaca mulatta; miRNA-Seq
library_construction_protocol: Previously dissected and frozen macaque brain tissue (weighing approximately 500 mg each) were dissected and treated with 20 units/ml papain (Worthington) in Hibernate A solution (5 ml/hemi-brain; BrainBits, Springfield, IL) and rocked for 15 min at 37° C. The brain tissue was gently homogenized in 10 ml/brain of cold Hibernate A solution. The brain homogenate was sequentially filtered through a 40 μm mesh filter (BD Biosciences), a 5 µm Filter (Pall Corporation) and a 0.2 μm syringe filter (Thermo Scientific). Exosomes were isolated from the filtrate as described previously. Briefly, the filtrate was sequentially centrifuged at 300 × g for 10 min at 4° C, 2000 × g for 10 min at 4° C, and 10,000 × g for 30 min at 4° C to discard cells, membranes, and debris. The supernatant was centrifuged at 100,000 × g for 60 min at 4° C to pellet exosomes. The exosome pellet was resuspended in 37 ml of cold PBS (Hyclone), and the exosome solution was centrifuged at 100,000 × g for 60 min at 4° C. The washed exosome pellet was resuspended in 2 mL of 0.95 M sucrose solution and inserted inside a sucrose step gradient column (six 2 ml steps starting from 2.0 M sucrose down to 0.25 M sucrose in 0.35 M increments, with the 0.95 M sucrose step containing the exosomes). The sucrose step gradient was centrifuged at 200,000 × g for 16 hr at 4° C. A 1 ml fraction was collected from the top of the gradient and discarded, and 6 mL of the gradient were collected in the exosome rich layers containing material with density higher then 1.07 (0.60 M sucrose layer) and lower than 1.17 (1.30 M sucrose layer) [28]. Pooled fractions were diluted to a volume 30 ml of cold PBS. 25 ml of this volume was taken for RNA extraction and 5 ml for Western blot studies. Sample volumes were brought up to appropriate volumes with cold PBS and centrifuged at 100,000 × g at 4° C for 60 min. PBS was pipetted off both pellets. Using the RNA isolated from exosomes, a small RNA library was generated using the Illumina Truseq Small RNA Preparation kit following the manufacturer's guidelines. The cDNA library was purified and used for cluster generation on Illumina's Cluster Station and then sequenced on the Illumina GAIIx. Raw sequencing reads were obtained using Illumina's Sequencing Control Studio software (version 2.8) following real-time sequencing image analysis and base-calling by Illumina's Real-Time Analysis (version 1.8.70). A pipeline script, ACGT101-miR v4.2 (LC Sciences), was used for sequencing data analysis. Sequences were then mapped to miRbase (version 20.0) [54]. 636 unique sequences mapped to both Macaca mullata mirs in miRbase and the Macaca mullata genome.
attributes.tag: GEO Accession
attributes.value: GSM2942584