GSM8194560: old_11; Homo sapiens; RNA-Seq
Cryopreserved human myocardium was powderised as described in the proteomic MS methods and weighed to approximately 30mg. Powderised myocardium was lysed and homogenised in 500 µL TRIzol (Invitrogen) using steel balls in the TissueLyser LT (Qiagen) and kept on dry ice between cycles (3-5 x 1 minute). 1-bromo-3-chloropopane (50 µL) was added to each sample and left to incubate for 5 minutes at RT, then spun at 14,000 x g at 4°C for 15 minutes to induce phase separation. RNA-containing aqueous phase was transferred to a sterile tube and an equal volume of isopropanol was added, mixed by inverting, and left for 1 hour at RT. RNA was pelleted at 14,000 x g at 18°C for 15 minutes, supernatant discarded. RNA was then washed in 4°C 70% ethanol twice discarding supernatant after spinning at 14,000 x g at 4°C for 10 minutes and left to dry at RT. RNA was DNase treated for 30 minutes at 37°C and washed again. Pelleted RNA was reconstituted in 15 µL 4°C nuclease free diethyl pyrocarbonate (DEPC). Concentration and quality were tested on NanoDrop (Thermo Fisher Scientific) to a standard of 260nm/280nm = 1.8-2.0 and 260nm/230nm 1.7-2.2. RNA integrity was assessed using an RNA Nano Chip on an Bioanalyzer (Agilent). RNA-seq libraries were prepared with Illumina Stranded Total RNA prep Ligation with Ribo Zero Plus (100ng input and 11 PCR cycles) according to manufacturer's instructions. Quality checks were performed using the Qubit dsDNA High Sensitivity Assay Kit and the Revvity LabChip GX Touch. The final sequencing pool was quantified using the Qubit dsDNA High Sensitivity Assay Kit after pooling all libraries equimolar into a single library pool. Sizing was checked using the Agilent High Sensitivity D1000 Tapestation system. The RNA-seq libraries were sequenced using a paired-end 250bp kit on a S4 flow cell of the Illumina NovaSeq 6000 with a final run concentration of 58pM and 1% PhiX.