GSM6703879: NIH3T3_no_click 2; Mus musculus; RNA-Seq
In order to harvest enough S phase cells, NIH3T3 cells were synchronized by 2 mM thymidine for 15 hours, followed by fresh medium culture for 2.5 hours. For each sample, 108 synchronized NIH3T3 or normal cultured mESCs were incubated with 10 mM EdU for 10 minutes, followed by PBS wash and hydroxyurea (HU) treatment.Next, the cells were fixed with 1% formaldehyde and then quenched with 0.125 M glycine. The cells were scraped, and permeabilized by ice-cold 0.25% Triton X-100/PBS for 30 minutes at room temperature. After washing with 0.5% BSA/PBS, the cells were re-suspended in click reaction buffer including 10 μM Biotin-azide or DMSO (negative control) for 1 hour at room temperature. After the click reaction, cells were washed with 0.5% BSA/PBS for three times, and re-suspended in lysis buffer (50 mM Tris-HCl, pH 8.0; 1% SDS; 1 μg/mL aprotinin; 1 μg/mL leupeptin) for sonication (30 seconds pulse/30 seconds pause, 60 cycles) using BioruptorTM UCD-200 machine. Finally, samples were centrifuged at 10000×g for 10 minutes at 4°C to remove the precipitate. The samples were incubated with streptavidin magnetic beads to purify replicaiton fork components. then the replicaiton fork components RNA was harvested using Trizol reagent.To avoid RNA degradation, all the buffers and reagents were prepared using DEPC-treated ddH2O and RNase inhibitors in the whole procedure. RNA libraries were prepared for sequencing using standard Illumina protocols