GSM5208221: mESC_SLAM-seq_DMSO_rep3; Mus musculus; OTHER
The SLAM-SEQ protocol was carried out as described in the SLAM-Seq Anabolic Kinetics Kit user guide (Lexogen #061.24) with modifications. Briefly, mESCs were seeded in 0.1% bovine gelatin (Sigma #G9391-500g) coated 6-well plates approximately two days prior to S4U labelling in order to attain an optimum confluency of around 70-80% (~2 x 106 cells). On the day of labelling, mESC media was aspirated and refreshed with pre-warmed mESC growth media containing either 500 nM dTAG-13 or DMSO to pre-establish acute degradation of BRD4. Time course for BRD4 degradation was set at 4.5 hours. Fifteen minutes into the time course, newly synthesized RNA was labelled via the addition of 4-thiouridine to all wells, including controls, at a final concentration of 500 µM/well and metabolic labelling was continued until the end of the time course. For the 4.5 hour time point, S4U medium was refreshed after 3 hours to continue efficient labelling. At the respective times points, media was aspirated and mESC cells were harvested following the addition of 400 µl of Accutase. The single cell suspension from each well was transferred into corresponding tubes and centrifuged at 2500 rpm for 5 minutes to pellet cells. The cell pellet was washed twice with ice cold 1x PBS and RNA extraction was carried out using the RNAeasy Plus kit (Qiagen #74134). Recommended amount of reducing agent (as indicated in the manufacturers' protocol) was added to all extraction buffers to maintain a reducing environment during extraction. 8 µg of total RNA was alkylated via treatment with 10 mM iodoacetamide (IAA) at 50°C for 5 minutes under optimal reaction conditions and purified by ethanol precipitation. IAA fixed RNA concentrations were estimated using the Nanodrop and approximately ~300 ng/µl of alkylated RNA per sample was sent to Lexogen for downstream processing and sequencing. Construction of SLAM-Seq libraries for sequencing on the Illumina NextSeq 500 platform was carried out using the Quant Seq 3' mRNA-Seq Library Prep Kit FWD for Illumina (Lexogen #015) was carried out at Lexogen GmbH according to the manufacturers' instructions.