title: Illumina HiSeq 3000 sequencing; Pro-seq of shControl and shRUNX1-ETO Kasumi-1 cells
design: Pro-seq of shControl and shRUNX1-ETO Kasumi-1 cells
library_construction_protocol: 25 million Kasumi-1 cells were collected 48h after lentiviral transduction for RUNX1-ETO knockdown. 25 million Kasumi-1 cells were collected 48h after lentiviral transduction for RUNX1-ETO knockdown and resuspended in cold swelling buffer followed by Dounce homogenization. Cells were centrifuged and washed with lysis buffer. Nuclei were centrifuged and washed again with lysis buffer followed by freezing buffer and stored at -80°C. Nuclear run-on (NRO) assays were performed with 2 biotin-11-NTPs (biotin-11-CTP and biotin-11-UTP, Jena Bioscience) as described in D. B. Mahat and colleagues (Nat Protoc, 2016). Briefly, nuclei were thawed on ice while the NRO mix was pre-equilibrated at 30°C. NRO was performed at 30°C, terminated with the addition of Trizol-LS (Ambion) and followed by RNA nuclear extraction. Nuclear RNA was fragmented by base hydrolysis and enriched for biotin-labelled RNA with Streptavidin beads (Dynabeads M280 Streptavidin, InVitrogen). Nascent biotinylated RNA was ligated with 3'-VRA RNA adaptor (5'Phos-GAUCGUCGGACUGUAGAACUCUGAAC-3'invdT). Ligated RNA was purified by a second round of Streptavidin beads enrichment and ligated with 5'-VRA RNA adaptor (5'-CCUUGGCACCCGAGAAUUCCA-3'). Ligated RNA was purified by a third round of Streptavidin beads enrichment followed by a reverse transcription step using SuperScript III First-strand synthesis (Invitrogen).  TruSeq small RNA kit