GSM7158601: TTseq_K562_REP1_UT_5min; Homo sapiens; OTHER
K562 cells were centrifuged (500g at 4°C for 5 minutes) and resuspended in TRIzol (5mL). One-fifth volume of chloroform was added to lysates, followed by shaking, incubation at room temperature for 2 minutes and centrifugation (13000g at 4°C for 10 minutes). The aqueous phase was isolated and RNA was precipitated using 100% isopropanol (1 volume), incubated at room temperature for 10 minutes and centrifuged (13000rpm at 4°C for 10 minutes). Supernatant was removed and RNA pellet was washed in 70% (v/v) ethanol, reconstituted in Ultrapure DNAse/RNAse-free distilled water (100μL) and denatured at 65°C for 10 minutes. S2 spike-in (15μg) was added to RNA (150μg), material was adjusted to a final volume 100μL and sonicated in microTUBE AFA Fiber screw cap tubes using the Covaris S220 Focused-ultrasonicator at a maximum power for 15 seconds. Thiol-specific biotinylation of RNA was performed in a final volume of 1.5mL by incubation with 10 mM tris (pH 7.4), 1 mM EDTA, 20% (v/v) dimethylformamide (200 μg/ml), and 300 μg of EZ-Link HPDP-Biotin for 1.5 hours at room temperature. An equal volume of chloroform was added to reaction, followed by shaking, incubation at room temperature for 2 minutes and centrifugation (500g at 4°C for 5 minutes). Aqueous phase was isolated and an equal volume of chloroform was added, followed by shaking, incubation at room temperature for 2 minutes and centrifugation (500g at 4°C for 5 minutes). This step was repeated an additional one time. Aqueous phase was isolated and RNA was precipitated using 5M NaCl (10% volume) and 100% isopropanol (1 volume) and centrifugation (20000g at 4°C for 20 minutes). RNA pellets were washed in 75% (v/v) ethanol, reconstituted in Ultrapure DNAse/RNAse-free distilled water (100μL) and denatured at 65°C for 10 minutes. Biotinylated RNA was separated from the total RNA pool by incubation with streptavidin beads at room temperature for 15 min with constant rotation. μMACs columns pre-equilibrated with room temperature wash buffer (100 mM tris-HCl (pH 7.4), 10 mM EDTA, 1 M NaCl, and 0.1% (v/v) Tween 20) were used to bind streptavidin beads, which were then washed using 900μL wash buffer that was heated to 65 °C or at room temperature, 5 times each. Biotinylated RNA was eluted into 700μL Buffer RLT from the RNeasy MinElute Cleanup Kit through two additions of 100 mM DTT (100μl) 3 min apart, and then isolated using the same kit according to manufacturer's instructions. RNA concentration was quantified using the Agilent 2200 TapeStation System with High Sensitivity RNA ScreenTape and Sample Buffer. NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (without additional fragmentation), where ribodepletion was performed using the NEBNext rRNA Depletion Kit. Total RNA-seq