GSM5217478: HDF161_Quiescent_rep_2; Homo sapiens; ncRNA-Seq
total RNA was isolated using TRI Reagent (Sigma Aldrich, Cat# T9424) following the manufacturer's protocol. 5 μg total RNA was hydrolyzed in 50 mM Carbonate-Bicarbonate buffer pH 9.2 and 10 mM MgCl2 at 95°C for 5 min, then put on ice. RNA cleavage was stopped by addition of 2 μl of 0.5 M EDTA pH 8.0. The RNA was precipitated by addition of 1/10 volume of 3 M sodium acetate and 2 volumes of 100% ice-cold EtOH, washed with 70% ice-cold EtOH and resuspended in nuclease-free water. RNA fragments were separated on a denaturing 6% acryl.:bis (19:1), 8 M urea gel. The fragments in the size range of 20 to 40 nt were extracted from the gel. The 5´ ends of the RNA fragments were dephosphorylated with Shrimp Alkaline Phosphatase (rSAP, New England Biolabs Cat# M0371S) as described in the manufacturer's protocol. The sequencing library was prepared using NEB SmallRNA for Illumina Kit (New England Biolabs) and sequenced as single-end reads in two technical replicas small RNA RNA-Seq NEB for Illumina)