title: GSM3066250: G08S01Y: Young 11; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA was isolated from 200 μL of serum from 13 young (30.9 ± 0.60 yrs) HANDLS participants and 10 old (81.8 ± 1.87 yrs) BLSA participants using TRIzol LS (Life Technologies, Waltham, MA) according to the manufacturer's instructions and with the addition of a second phenol/chloroform extraction prior to RNA precipitation as previously described (Noren Hooten et al. 2013). RNA samples were resuspended in 20 μL of RNAse-free water and frozen at -80°C until further use. RNA quality was assessed using an Agilent BioAnalyzer (Agilent Technologies, Santa Clara, CA). Total RNA was fragmented to <200 bp using the Bioruptor NGS (Diagenode, Denville, NJ). Briefly, RNA was diluted in EB buffer to a final volume of 100 µl and subjected to bioruption for 30 min (Power: H position) in an ice-cold DI water bath using 30”/30” ON/OFF cycles with cold water bath replacement every 7.5 min. Sample RNA was then dry vacuumed to 20 μL and tested for fragmentation using the Agilent BioAnalyzer Small RNA chip. Total RNA isolated as described above was prepared for sequencing using the Ion Total RNA-seq Kit v2 (Life Technologies) with multiplexing according to the manufacturer's instructions.  The Life Technologies Ion RNA Adaptors were added to the fragmented RNA at 65oC for 10 min and then allowed to hybridize at 30oC for 5 min.  A ligation reaction was done at 30oC for 1 hr.  The Life Technologies reverse transcription primer was incubated with the ligated samples at 70oC followed by snap-cooling on ice for hybridization. A reverse transcription reaction was done using Superscript III at 42oC for 30 min. Clean-up of the cDNA was done using the supplied magnetic bead cleanup module and 100% ethanol. Samples were eluted from the beads using 37oC nuclease free water. Amplification and addition of sample specific indexes was performed using 16 cycles of PCR to enrich the samples for fragments that contained adaptors on both ends, followed by another Magnetic bead clean up.  These enriched samples were tested using the Agilent Bio-Analyzer High Sensitivity DNA chips. Barcoded samples were pooled, and each library template was clonally amplified on Ion Sphere™ Particles in the Ion One Touch II for sequencing on the Ion Proton™ System.