GSM7850293: TNF_MSC_2; Homo sapiens; RNA-Seq
MSCs were grown to confluence in 75-cm2 filter cap cell culture flasks, washed with PBS, and the media was replaced with StemPro MSC serum-free media (SFM) CTS (Gibco Life Technologies, Gaithersburg, MD). Cells were incubated 18-24 hours upon which the conditioned culture media (CM) was collected and exosomes were isolated by differential ultracentrifugation essentially as previously described.21  The CM was centrifuged using a Beckman Coulter Allegra® X-15R centrifuge (2000 g at 4°C for 20 minutes) to remove any detached cells, apoptotic bodies, and cell debris. Clarified supernatant CM was then centrifuged in a Beckman Coulter Optima™ L-80XP Ultracentrifuge (100,000 g at 4°C for 2 hours) with a SW 28 rotor to pellet exosomes. The supernatant was carefully removed, and exosome pellets were re-suspended in PBS and pooled. Exosomes were characterized using a Thermo NanoDrop spectrophotometer for protein and RNA concentration. Particle diameter and concentration were evaluated via IZON qNano Nanoparticle Characterization instrument (Zen-bio Inc, Research Triangle Park, NC). Total RNAs were isolated from Human MSCs and MSCs-derived exosomes, as well as rat medial collateral ligaments (MCLs) (post-injury day 14) using trizol (ThermoFisher #15596018) and chloroform phase separations followed by the RNeasy mini protocol (Qiagen #74106) with optional on-column Dnase digestion (Qiagen #79254). One hundred nanograms of total RNA were used to prepare sequencing libraries using the LM-Seq (Ligation Mediated Sequencing) protocol mRNA-seq: RNAs were selected using the NEB Next Poly A+ Isolation Kit (NEB #E7490S/L).  Poly A+ fractions were eluted, primed, and fragmented for 7 minutes at 85°C.  First stand cDNA synthesis was performed using SmartScribe Reverse Transcriptase (Takara Bio USA #639538), and RNA was removed.  cDNA fragments were purified with Ampure XP beads (Beckman Coulter #A63881). The 5' adapter was ligated, and 18 cycles of amplification were performed. These final indexed cDNA libraries were quantified, normalized, multiplexed, and run as single-end reads on the HiSeq 3000; small RNA-seq: Total RNAs were isolated using trizol (ThermoFisher #15596018) and chloroform phase separations followed by the RNeasy mini protocol (Qiagen #74106) with optional on-column DNase digestion (Qiagen #79254). One hundred nanograms of total RNA was used to prepare sequencing libraries using the NEBNext Multiplex Small RNA Library Prep Set for Illumina (NEB #E7560S). The 3' adapter was ligated and the reverse transcription primer hybridized.  The 5' adapter was ligated and cDNA synthesis was performed using ProtoScript II Reverse Transcriptase.  PCR amplification was performed with 15 cycles and purified with Qiaquick PCR Purification Kit (Qiagen # 28106).  cDNA libraries were then size-selected using AMpure XP beads (Beckman Coulter #A63881).