title: GSM9102874: SY5Y_nuclear_rep2; Homo sapiens; RNA-Seq
library_construction_protocol: Nuclear extraction: Nuclear isolation from total cells was performed following a previously established protocol(Nabbi and Riabowol 2015). Briefly, trypsinized cells were pelleted, washed with ice cold 1X PBS, and resuspended in ice cold Nuclear Isolation Buffer (10mM Tris-HCl, pH 7.4, 10mM NaCl, 3mM MgCl2, 0.3% NP40, Roche EDTA-free protease inhibitor (Millipore Sigma, 11836153001), Sigma PhosSTOP phosphatase inhibitor (Millipore Sigma, 4906845001). The suspension was briefly centrifuged again to pellet the nuclei. The supernatant was removed (cytoplasmic fraction) and the pellet was resuspended in ice cold Nuclear Isolation Buffer. The suspension was centrifuged at 10,000 RCF and supernatant removed. The nuclear pellet was resuspended in TRIzol (Invitrogen, 15596018) before RNA isolation. Nascent RNA isolation from nuclei: Cells were incubated with 1mM 4-thiouridine (Sigma, T4509), dissolved in wter, for 8 minutes and spun down to pellet cells. The pellet was resuspended in Trizol and flash frozen in liquid nitrogen. After nuclear isolation and RNA isolation and described below, 4sU pulldowns were performed using MTSEA biotin-XX (VWR, 89139-636) 4sU-containing (nascent) transcripts were purified following previously established protocols(Garibaldi et al. 2017; Duffy and Simon 2016).  RNA was extracted from whole cells or nuclear pellets using TRIzol reagent to separate the aqueous RNA phase from the DNA and protein phases. The aqueous RNA phase was carried forward into column purification using a Zymo-25 RNA Clean & Concentrator kit (Zymo, R1018) Library preparation for polyA RNA, CHX-treated, NMDi14-treated, and DMSO-treated samples was performed using the KAPA Stranded mRNA-Seq Kit for Illumina Platforms (KK8421). Library preparation for the nuclear and 4sU-labeled samples was performed using Kapa Biosystems KAPA RNA HyperPrep Kit with RiboErase (KK8561). Libraries were sequenced commercially at Novogene using an Illumina Novaseq 6000,  Illumina Nextseq 2000. SIRV libraries were made with both KAPA Stranded mRNA-Seq Kit and KAPA RNA HyperPrep Kit and sequenced on Illumina NextSeq 500