Transcriptomic Modulation by Exosomes Derived from Human Adipose Stem Cells in Neuronal Cells
Transcriptomic Modulation by Exosomes Derived from Human Adipose Stem Cells in Neuronal Cells
The cells were treated with different conditions for 24 h and collected for RNA extraction. Total RNA was extracted according to the instruction manual of TRlzol Reagent (Life Technologies, California, USA). The RNA concentration and purity were measured via a NanoDrop 2000 (Thermo Fisher Scientific, Wilmington, DE). RNA integrity was assessed via both agarose electrophoresis and the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA).  A total of 1 μg of RNA per sample was used as input material for the RNA sample preparations. The sequencing libraries were generated via the NEBNext UltraTM RNA Library Prep Kit for Illumina (NEB, USA) following the manufacturer's recommendations, and index codes were added to attribute the sequences to each sample. Briefly, mRNA was purified from total RNA via poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First-strand cDNA was synthesized via random hexamer primers and M-MuLV reverse transcriptase. Second-strand cDNA synthesis was subsequently performed via DNA polymerase I and RNase H. The remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of the 3' ends of the DNA fragments, NEBNext adaptors with hairpin loop structures were ligated to prepare for hybridization. To preferentially select cDNA fragments 240 bp in length, the library fragments were purified with the AMPure XP system (Beckman Coulter, Beverly, USA). Then, 3 μl of USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min, followed by 5 min at 95°C before PCR. Then, PCR was performed with Phusion High-Fidelity DNA polymerase, universal PCR primers and Index (X) primers. Finally, the PCR products were purified (AMPure XP system), and library quality was assessed on an Agilent Bioanalyzer 2100 system.