GSM2575923: vESRD_NE44; Homo sapiens; RNA-Seq
EDTA-treated whole blood samples from individual study subjects were centrifuged at 1,600 g for 10 min at 4 °C, transferred to a new centrifuge tube and centrifuged again at 16,000 g for 10 min to remove any residual blood cells. Plasma samples were stored at -80 °C until the step of RNA extraction. Six ml Trizol LS was mixed with 2 ml plasma sample for 5 min at room temperature (RT), followed by mixing with 1 ml 1-bromo-3-chloropropane, vortexed and incubated at 4 °C for 10 min, centrifuged at 6,000 g for 30 min for phase separation. The aqueous phase layer (~ 4 ml) was transferred into a new 50 ml centrifuge tube, mixed with 40 ul glycogen and 5 ml isopropanol, incubated at -20°C for 15 min, centrifuged at 6,000 g for 30 min at 4 °C. After removal of the supernatant, the pellet was lysed with the mixture of Lysis Solution (300 ml) and miRNA Homogenate Additive (30ml) from mirVana (Thermo Fisher, AM1650) kit, vortexed and fully resuspended, followed by addition of 350 ml isopropanol. The mixture was transferred onto an RNAqueous micro filter cartridge and centrifuged at 10,000 g for 15 sec, washed with 700 ml Wash Solution 1 and centrifuged at 10,000 g for 10 sec, washed again with 500 ml Wash Solution 2/3 and centrifuged at 10,000 g for 1 min. Eight ml of Elution Solution (preheated to 65°C) was applied to the filter and centrifuged 10 sec at 10,000 g to obtain RNA samples. The final step was repeated again and the RNA samples were mixed, quantified using Qubit RNA HS assay kit and stored at -80 °C until RNA sequencing. (Exploratory cohort) For the plasma transcriptome RNASeq in the exploratory cohort, 10 ng of total plasma RNA samples from individual study subjects was reversed transcribed to cDNA followed by PCR amplification (16 cycles), digestion with FuPa enzyme, ligation (60 minutes) and Ampure cleanup according to the user guide supplied with the Ion AmpliSeq Transcriptome Human Gene Expression Kit (Thermo Fisher Scientific, PN A26325).  Barcoded libraries were quantitated by Ion Library TaqMan™ Quantitation Kit (Thermo Fisher Scientific, PN4468802) and combined to either 7- or 8- plex pools for sequencing. Sequencing template preparation and Proton P1 chip loading were performed on the Ion Chef™ instrument using the Ion Proton™ Hi-Q ™ Chef kit. (Validation cohort) For the plasma transcriptome RNASeq in the validation cohort, 10 ng of total plasma RNA samples from individual study subjects was reversed transcribed to cDNA followed by PCR amplification (16 cycles) using predesigned primers targeting 8 candidate lncRNAs and 6 control mRNAs, digestion with FuPa enzyme, ligation (60 minutes) and Ampure cleanup.  Barcoded libraries were quantitated by Ion Library TaqMan™ Quantitation Kit (Thermo Fisher Scientific, PN4468802) and pooled for sequencing. Sequencing template preparation and Proton P1 chip loading were performed on the Ion Chef™ instrument using the Ion Proton™ Hi-Q ™ Chef kit.