GSM8223054: LDB1_AID_untreated_TT-seq_rep1; Mus musculus; OTHER
Exponentially growing cells in T-150 flasks were labeled with 500 μM 4-thiouridine (4SU) (MedChemExpress), for 5 minutes. The cells were processed with 2 mL TRIzol Reagent (Invitrogen) for every 10 million G1E-ER4 cells and total RNAs was extracted following the user manual. 500 ng of 4SU-labeled Drosophila Schneider 2 (S2) cells total RNA as spike in was mixed with 100 μg of collected 4SU-labeled G1E-ER4 total RNA. Mixed RNA was fragmentated using a final concentration of 0.2 M NaOH for 18 minutes and neutralized with a final concentration of 0.5 M Tris-HCl (pH 6.8). RNA was purified by isopropanol precipitation. Labelled RNA was biotinylated in 300 μL of biotinylation mix (fragmented total RNA, 10 mM HEPES pH 7.5, 1 mM EDTA, 0.167 mg/mL MTSEA-biotin (Biotium)) for 1 hour at room temperature and purified with phenol/chloroform/isoamyl alcohol (25:24:1 (vol/vol/vol)) extraction. Denature the biotinylated RNA from at 65 °C for 10 minutes, followed by rapid cooling on ice for 5 minutes. The denatured biotinylated RNA was bound with Dynabeads MyOne Streptavidin C1 (Invitrogen) at room temperature for 30 minutes, eluted with 100 mM DTT and subsequently purified by isopropanol precipitation. Quality of total RNA extraction, RNA fragmentation (we aimed for an RNA size distribution between 25 and 500 nt), and purified 4SU-labeled RNA were determined using Agilent TapeStation RNA ScreenTape (Agilent). Strand-specific sequencing libraries were generated with the Illumina Stranded Total RNA Prep (Illumina) and IDT for Illumina RNA UD Indexes Set A, Ligation (Illumina). Library size was determined using Agilent TapeStation High Sensitivity DNA ScreenTape (Agilent).