title: GSM1186445: V6.5_5minFP_#2; Mus musculus; OTHER
library_construction_protocol: Nuclei isolation are performed using standard protocols, in brief, after rinsing the 15 cm2 plates with drug-treated cells, 15 ml cell lysis buffer (10 mM Tris-Cl, pH 7.5, 300 mM Sucrose, 3mM CaCl2, 2 mM MgAc2, 0.5% NP-40, 5 mM DTT, 1 mM PMSF, protease inhibitors) was added to the plates and the cells were scraped off, and spun down at 4°C and 185 xg for 5 min in a GS-6R Beckman swing-bucket centrifuge, after which supernatant was discarded. 5 ml fresh cell lysis buffer was added and cells were dounced 50 times in a 5 ml douncer on ice and spun down at 328 xg for 5 min, after which supernatant was discarded and nuclei were taken up in ~250 μl of glycerol storage buffer (50 mM Tris-Cl, pH 8.3, 40% glycerol, 0.1 mM EDTA, 5 mM MgAc2, 5 mM DTT, 1 mM PMSF, protease inhibitors) and snap frozen. For each nuclear run-on (NRO), 1e7 nuclei were used. An NRO master mix was added containing nucleotides ATP, GTP, CTP and nucleotide analogue bromo-UTP, as well as sarkosyl and 300 mM KCl to remove all impediments that pause Pol II and prevent NRO and to prevent re-initiation of Pol II. The NRO was performed at 37°C for 5 min, which results in ~100 nt addition to the nascent RNA. Immediately after the NRO, a population of ~100 different in-vitro transcribed Arabidopsis thaliana spike-in RNAs with and without Br-UTP was added to the nascent RNA as a way to assess experimental differences between each library, and to use as a normalization tool between timepoints within each timecourse.  The nascent RNA was fragmented to ~150 nts and NRO BrU-RNA was isolated 3 consecutive times with BrdU-antibody beads (sc-32323, Santa Cruz), with enzymatic TAP and PNK treatments to remove the cap and 3’-phosphate and to add a 5’-phosphate, as well as Illumina adaptor ligations between the BrU-RNA isolation steps. The three consecutive isolation steps lead to an approximate 500.000x enrichment of BrU-RNA over background RNA. BrU-RNA was reverse transcribed, amplified, barcoded and sent for sequencing. Each dataset was done in replicate.
attributes.tag: GEO Accession
attributes.value: GSM1186445