GSM2611288: Fbxl19fl_72hrTAM_rep3; Mus musculus; RNA-Seq
For labelling of nascent RNA transcripts, cells were grown in 15cm culture dishes. Labelling was performed for 20 minutes at 37°C by the addition of 50mM 4-thiouridine (T4509, Sigma) to the culture medium. After the incubation, the medium was removed and RNA was isolated using TRIzol reagent (Thermo scientific) following manufacturer’s instructions. The total RNA was treated with TURBO DNA-free kit (Ambion, Thermo scientific) in order to remove contaminating genomic DNA. 300µg RNA was biotinylated using 600 µg Biotin-HPDP (21341, Pierce, Thermo scientific) in Biotinylation buffer (100mM Tris-HCl pH 7.4, 10mM EDTA). The reaction was carried out for one and half hour on a rotor at room temperature. Unincorporated Biotin-HPDP was removed by chloroform/isoamylalcohol (24:1, Sigma) wash followed by isopropanol precipitation of the biotinylated RNA. Labelled biotinylated RNA was isolated using µMacs Streptavidin Kit (130-074-101, Miltenyi) following manufacturer’s instructions and purified using RNeasy MinElute Cleanup kit (Qiagen). The quality of the RNA was confirmed using the RNA pico Bioanalyser kit (Agilent). Up to 1µg purified 4sU-labelled RNA was used to prepare libraries for 4sU-seq. Ribosomal RNA was removed using NEBNext® rRNA Depletion Kit and libraries were prepared using NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina (NEB) following manufacturer’s instructions. Library quality was assessed using the High-sensitivity DS DNA Bioanalyser kit (Agilent) and the concentration was measured by quantitative PCR using KAPA Library quantification standards for Illumina (KAPA Bioasystems). All RNA-seq experiments were carried out in biological triplicates. RNA-seq libraries were sequenced as 76 paired-end reads on Illumina NextSeq500 platform using NextSeq® 500/550 High Output Kit v2 (150 cycles).