Extracellular Vesicle P100 Fraction Small RNAs

Extracellular Vesicles were isolated from primary cultures of neural stem cells (NSCs) and astrocytes by performing serial centrifugation and a 100,000 x g ultracentrifugation step. The goal of this experiment is to compare EV small RNAs from two cell types. Overall design: EVs were isolated by serial centrifugation from primary neural stem cells (NSCs) cultured from the subventricular zone or cortical astrocytes of CD1 mice. Small RNA libraries were constructed with the CleanTag Small RNA Library Preparation Kit (TriLink,Cat# L-3206) according to the manufacturer's protocol. The final purified library was quantified with High Sensitivity DNA Reagents (Agilent Technologies, PO# G2933-85004) and High Sensitivity DNA Chips (Agilent Technologies, PO# 5067-4626). The libraries were pooled, and the 140bp to 300bp region was size selected on a 8% TBE gel (Invitrogen by Life Technologies, Ref# EC6215). The size selected library is quantified with High Sensitivity DNA 1000 Screen Tape (Agilent Technologies, PO #5067-5584), High Sensitivity D1000 reagents (Agilent Technologies, PO# 5067-5585), and the TailorMix HT1 qPCR assay (SeqMatic, Cat# TM-505), followed by a NextSeq High Output single-end sequencing run at SR75 using NextSeq 500/550 High Output v2 kit (Cat #FC-404-2005, Illumina, San Diego, CA) according to the manufacturer's instructions.