title: GSM5235637: ZB318_cycling; Mus musculus; OTHER
library_construction_protocol: Briefly, 10 million cells were collected and permeabilized with the buffer (10 mM Tris-HCl pH 7.4, 300 mM sucrose, 10 mM KCl, 5 mM MgCl2, 1 mM EGTA, 0.05% Tween-20, 0.1% NP40 substitute, 0.5 mM DTT, protease inhibitors and Rnase inhibitor). The permeabilized cells were resuspended in 100 ml of storage buffer (10 mM Tris-HCl pH 8.0, 25% (V/V) glycerol, 5 mM MgCl2, 0.1 mM EDTA and 5 mM DTT) for nuclear run-on with 2X run-on mix (5 mM Tris-HCl PH 8.0, 2.5 mM MgCl2, 0.5 mM DTT, 150 mM KCl, 0.5 mM ATP, 0.5 mM CTP, 0.5 mM GTP, 0.5 mM BrUTP, RNase inhibitor, 1% sarkosyl) at 37o for 5 min. RNA was extracted by Trizol and followed by hybrolyzation with NaOH at a final concentration of 0.2 N on ice for 18 min. After quenching with ice-cold Tris-HCl PH6.8 at a final concentration of 0.55 M and exchanging buffer via Bio-Rad P30 columns, the RNA was incubated with Br-dU antibody-conjugated beads (Santa Cruz biotechnology, sc-32323-ac) for 1 hr. The enriched run-on samples were incubated with RppH (NEB, M0356S) and hydroxyl repair with T4 PNK (NEB, M0201S), followed by ligating the 5' and 3' RNA adaptor. RT-PCR was performed from the adaptor-ligated RNA to obtain cDNA Half of the cDNA was subjected to making Groseq libraries by two rounds of PCR with barcode primers.  200-500 bp products from the first round of PCR were subjected to the second round of PCR with the number of PCR cycles determined by test PCR amplification. The second round of PCR products were size-selected by SPRIselect beads (Beckman Coulter, B23318).