title: GSM4292522: S76_3hPost; Homo sapiens; miRNA-Seq
library_construction_protocol: For exosomal RNA isolation, 700 µl of Qiazol (Qiagen, Germantown, MD, USA) were added to 50 µl of exosome preparation, and the mixture was incubated for 5 min at room temperature. After the addition of 140 µl of chloroform, the mixture was vortexed for 10 sec and incubated for 3 min at room temperature. After the upper aqueous phase was transferred to a fresh tube, a two-fold volume of absolute ethanol (Sigma-Aldrich, St. Louis, US) was added, and the mixture was vortexed briefly. Samples were then transferred to RNeasy mini spin columns (Qiagen) that were centrifuged at 10,000 x g for 30 sec at 4ºC, and flow-through was discarded. An 80-µl volume of DNase incubation mix was added directly onto the column membrane and samples were incubated for 15 min at room temperature. Following wash steps with RWT buffer, RPE buffer (Qiagen), and 85% ethanol, RNA pellets were dried by centrifugation at 10,000 x g for 1 min, and eluted in 15 µl of water. Small RNA libraries were prepared from exosome-derived RNA using the NEB Next Small RNA Library Prep Set for Illumina (New England Biolabs, Ipswich, MA, USA), according to the manufacturer's instructions. Briefly, RNA-Seq libraries were constructed using 2 ng of exosomal RNA and 15 cycles of PCR amplification. Libraries were run on a 6% TBE gel for 60 min at 120 V, and bands between 140 to 160 bp were excised from the gel using a razor blade. Gel pieces were crushed into smaller pieces and incubated in water overnight on a rotator. Libraries were isolated using gel filtration columns. Following ethanol precipitation, the resulting pellets were each resuspended in 11 µl of ultrapure water. All libraries were analyzed on an Agilent 2100 Bioanalyzer using the Agilent High Sensitivity DNA kit. Small RNA-Seq; for sequencing, libraries were pooled at equimolar concentrations, mixed with 5% PhiX (Illumina, San Diego, CA, USA), and single-end sequencing was performed on an Illumina MiSeq platform following the manufacturer's recommendations.