title: GSM5508645: HOGD0 2; Homo sapiens; miRNA-Seq
library_construction_protocol: Cultured cells were harvested then centrifuged at 1,500 g and cell pellets were used for RNA isolation. Cell pellet was homogenized in TRIzol using a hand-held pestle homogenizer and incubated in TRIzol for at least 5 minutes. Chloroform (1:5 ratio) was added, mixed well and incubated at room temperature for 15 minutes. Samples were then centrifuged at 12,000 g for 15 minutes at 4°C. The top aqueous layer was transferred to a clean tube, and the RNA was precipitated in 3 M NaAc pH 5.2 (10:1 ratio), 4 ml of glycogen (5 mg/ml), 100% isopropanol (1:1 ratio) overnight at -80°C. The next day, the samples were centrifuged at 20,000 g for 20 minutes at 4°C. The resulting RNA pellet was washed once in 75% ethanol, centrifuged at 7,500 g for 10 minutes at 4°C. The washed RNA pellet was dissolved in nuclease-free water. RNA was quantified by NanoDrop and the quality was confirmed by agarose gel. For small RNA-seq library, total RNA sample quality was assessed by RNA ScreenTape (Agilent Technologies Inc., California, USA) and quantified by Qubit 2.0 RNA HS assay (ThermoFisher, Massachusetts, USA). All library construction was prepared according to the QIAseq miRNA Library (Qiagen, Hilden, Germany) per manufacturer's instructions. Final libraries quantity was assessed by Qubit 2.0 (ThermoFisher, Massachusetts, USA) and quality was assessed by TapeStation HSD1000 ScreenTape (Agilent Technologies Inc., California, USA). Final library size was about 180 bp with an insert size of about 20 bp. Illumina® 8-nt single-indices were used. Equimolar pooling of libraries was performed based on QC values and sequenced on an Illumina® HiSeq (Illumina, California, USA) with a read length configuration of 150 PE for 10M Single-ended reads per sample.