GSM9341437: PROseq_SA1_KO_rep3; Mus musculus; OTHER
Briefly, 10 million cells were collected and permeabilized with permeabilization buffer. The permeabilized cells were resuspended in 100 ml of storage buffer for nuclear run-on with 2X run-on mix at 37℃ for 5 min. RNA was extracted by Trizol and followed by hybrolyzation. The RNA was incubated with streptavidin C1 beads (Thermo Fisher Scientific, #65001) and the enriched run-on samples were incubated with hydroxyl repair with T4 PNK (NEB, M0201S) and RppH (NEB, M0356S), followed by ligating the 5' and 3' RNA adaptor. RT-PCR was performed from the adaptor-ligated RNA to obtain cDNA. The cDNA was subjected to making PRO-seq 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). PRO-seq libraries were sequenced via single end 50bp or paired end 150bp sequencing on a Nova-seq 6000 (Illumina) sequencer and normalized to a coverage of 10 million 100nt reads for display.