title: Nascent RNA-seq data of mouse embryonic stem cells treated with auxin for CHD4 depletion against untreated control
design: Nascent RNA-seq data of mouse embryonic stem cells treated with auxin for CHD4 depletion against untreated control
library_construction_protocol: Cells were grown in 10cm plates. Upon aspiration of the medium, cells were harvested in 3ml Trizol Reagent (Ambion, 15596026). The cell lysate was homogenised by pipetting up and down several times and transferred into a tube. ES cells were cultured on 0.1% gelatin-coated plates in 2i/LIF conditions. For CHD4-mAID depletion, 0.5mM auxin (1:1000 from DMSO stock) was resuspended in pre-warmed culture media and transferred to the cells. Control cells were not treated with auxin (no auxin). Cells were treated with 0.3mM 4sU for 30min. Total RNA was extracted. Genomic DNA was removed using a DNase treatment step, and RNA was then purified by phenol:chloroform extraction and ethanol precipitation. The total RNA was fragmented using ultrasound sonication. Fragmented RNA was then subjected to biotinylation by incubating with HPDP-biotin at room temperature for 2 hours in the dark, allowing selective labelling of 4sU-containing transcripts. Unincorporated biotin was removed by repeated chloroform extraction, and the RNA was precipitated using ethanol. Biotinylated RNA was enriched using streptavidin-coated magnetic beads. The beads were washed to reduce background binding, and 4sU-labelled RNA was eluted using 100 mM DTT. Eluted RNA was purified using the miRNeasy micro kit (Qiagen, 217084). Libraries were prepared from the enriched nascent RNA using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina, following the manufacturerâ€™s protocol with slight modifications to accommodate low RNA input. Libraries were quantified using Qubit and quality-checked using the Agilent Bioanalyzer.