Solid 5500xl platform

Total RNA was extracted from serum-starved MSCs following treatment with 10 ng/mL recombinant human TGFα (PeproTech, Rocky Hill, NJ, USA) for 1 h, using the TRIzol Reagent (Invitrogen, Milan, Italy). The small RNA fraction was enriched using the PureLink miRNA Isolation Kit (Thermo Fisher Scientific, Milan, Italy). The quantity and quality of enriched samples were determined by the Agilent 2100 Bioanalyzer (Agilent Technologies, Milan, Italy). Then, samples were subjected to hybridization and ligation to SOLiD adaptor mix, using the SOLiD Small RNA library preparation protocol with the SOLiD Total RNA-Seq kit (Thermo Fisher Scientific). Libraries of cDNA were generated by reverse transcription and purified using the MinElute PCR Purification kit (Qiagen, Milan, Italy). Following size selection of cDNA, amplification was performed using SOLiD 5′PCR primers and barcoded SOLiD 3′PCR primers (Thermo Fisher Scientific). Amplified cDNA was purified using the PureLink PCR Micro Kit and quantified with the Qubit fluorometer (Thermo Fisher Scientific). Barcoded cDNA libraries were captured onto the surface of beads, amplified by emulsion PCR and enriched using the SOLiD EZ Beads System (Thermo Fisher Scientific). Beads were deposited onto glass slides and sequenced on the SOLiD 5500xl platform (Thermo Fisher Scientific) using the fragment protocol (35 bp).

PolyA-RNA was isolated from 25 μg of total RNA using the MicroPoly(A) Purist kit (Ambion, USA). Total PolyA-RNA was used to generate whole transcriptome libraries for sequencing on the SOLiD 5500XL platform following the manufacturer’s recommendations (Life Technologies; CA, USA). Amplified cDNA quality was analyzed using the Bioanalyzer 2100 DNA 1000 kit (Agilent Technologies; Spain) and quantified using the Qubit 2.0 Fluorometer (Invitrogen; UK). Whole transcriptome libraries were used to make SOLiD templated beads following the SOLiD templated bead preparation guide. This protocol consisted of an RNA enrichment and chemical modification step, followed by a clonal amplification step. Bead quality was estimated based on work flow analysis parameters. The samples were sequenced using the 50625 paired-end protocol, generating 115 nt sequences consisting of 75 nt plus 35 nt (Paired-End) + 5 nt (Barcode). Quality data was measured using software parameters of the SOLiD Experimental System.