GSM9322433: H9,Time1; Homo sapiens; ncRNA-Seq
The total RNA was extracted from 2.5 ml of whole blood samples by using a PAXgene Blood miRNA Kit (PreAnalytiX, Qiagen, Switzerland). The RNA quantity and concentration were measured with a Qubit 4.0 Fluorometer using a High-Sensitivity RNA kit, the Qubit® RNA HS (High Sensitivity) Assay Kit (both ThermoFisher Scientific, Wilmington, DE, USA). The RNA quality and fragmentation were assessed with a Fragment Analyzer using an SS RNA kit (both Agilent Technologies, Santa Clara, CA, USA). The mean RNA integrity number (RIN) for all samples was 7.6 (range: 4-9.6), with individual RINs of 8.6, 7.1, and 7 for sampling times T1 to T3, respectively. The following analysis steps were performed to identify a broad spectrum of individual RNAs, including mRNA, rRNA, tRNA, miRNA, snRNA, snoRNA, lncRNA, piRNA, other sRNAs, and unannotated and unmapped RNAs. RiboMarker® RNA Fragmentomics Library Preparation Kit #400-00012 (RealSeq Biosciences, Santa Cruz, USA) was used for preparing all individual libraries from 50 ng of the total RNA according to the manufacturer's protocol. Briefly, the two-day workflow involved: (i) RNA pretreatment [with RNA Clean & Concentrator-5 #R1015 (Zymo Research, Irvine, CA, USA)], adapter ligation, and blocking on the first day, followed by (ii) circularization, dimer removal, reverse transcription, PCR amplification, and size selection with SPRI beads on the second day. The RNA libraries concentration was measured with the 1× dsDNA HS kit (ThermoFisher Scientific) on the Qubit 4.0 fluorometer, and the quality was checked using the Fragment Analyzer with the HS NGS Fragment Kit (Agilent Technologies). The libraries had estimated profiles with the main peak of fragment length between 147-162bp.