GSM6481403: r1-MD6-contr; Homo sapiens; RNA-Seq
For the analysis of nascent RNA expression, cells were treated with RA as described above and pulse-labeled with 0.5 mM 5-ethynyluridine for 30 min at 37°C and 5% CO2. After pulse-labeling, total RNA was isolated using TRIzol and purified RNA was further processed using the Click-iTTM Nascent RNA Capture Kit (Invitrogen, C10365) In brief, 5-EU incorporated RNA was biotinylated, purified and pulled down using Dynabeads™ MyOne™ Streptavidin T1 beads. Before the pulldown, biotinylated custom-made spike-in RNAs #1 (2.5e-5 ug) and #2 (2.5-e-4 ug) (expression vectors kindly provided by Kenneth Zaret, University of Pennsylvania) were added to 1 ug of biotinylated RNA. After washing the beads, cDNA was directly generated off the beads using the Universal Plus Total RNA-Seq with NuQuant kit (Tecan) as described by the manufacturer with the following modifications: fragmentation time was increased to 10 min and after second strand synthesis, beads were collected on a magnet and supernatant was transferred to a new tube for subsequent steps. Quality of the libraries was assessed using the 2100 Bioanalyzer (Agilent cat. #G2939BA) and the Qubit fluorometer (Thermo Fisher cat. #Q33226), pooled and sequenced on an Illumina NovaSeq6000 with 150 bp PE. 