GSM8458113: oeCTCFwt_IAA_rep1; Mus musculus; OTHER
Exponentially growing cells were labeled with 400μM 4-thiouridine (4sU) for 15min at 37°C under 5% CO2. The cells were lysed in TRIzol and total RNA was extracted following TRIzol protocol. 100μg mixed RNA was fragmentated using NaOH (0.2M as a final concentration) for 20min and neutralized with Tris-HCl (pH 6.8) (0.5M as a final concentration) immediately followed by RNA purification withisopropanol precipitation. Labelled RNA was biotinylated at RT for 3 h and purified with chloroform extraction. The denatured biotinylated RNA was bound withDynabeads™ MyOne™ Streptavidin C1 at RT for 15 min, eluted with 100mM DTT and subsequently purified with RNA clean beads. Strand-specific libraries were generated with the VAHTS Universal V8 RNA-seq Library Prep Kit for Illumina.