GSM8516863: WT_Treated_8h_rep2_steady_state; Homo sapiens; RNA-Seq
To calculate changes in nascent RNA synthesis (TTseq): Parental (untagged), CNOT1AID and CNOT4AID cells were pretreated with 1mM auxin for 2 h, follwed by pulse labelling with 4-thiouridine (4SU) for 15 minutes, followed by total RNA extraction. Total RNA was extracted from the cells using Trizol (Invitrogen#15596026) and cleaned up using PureLink RNA Mini kit (Invitrogen#12183018A) For libraries to caluculate changes in steadystate and RNA halflife (Triptolide libraries), 500 ng of Turbo DNase digested RNA was mixed with 2 µl of 1:200 diluted ERCC Spike-in mix (Invitrogen#4456740) which was subjected to library preparation. TT-seq. libraries were prepared as described by Gregersen et al., 2020 (PMID: 31915390). Briefly, following depletion of desired protein, cells were treated with 4-thiouridine (4SU) for 15 minutes. The total RNA was extracted using Trizol and PureLink RNA Mini kit. RNA was mixed with that from S. pombe labelled with 4TU (as a spike in control). The labelled RNA was biotinylated using methanethiosulfonate (MTS)-biotin (Biotium#90065) and enriched using Dynabeads™ MyOne™ Streptavidin C1 (Invitrogen#65001). 300 ng of RNA was subjected to library preparation. All the sequencing libraries were prepared using Illumina Stranded Total RNA Prep with Ribo-Zero Plus (Illumina#20040525) according to manufacturer's instructions. Sequencing was carried out at Genomics Research Incubator, Penn State University, USA using NextSeq™1000/2000 P2 Reagents(100Cycles) on NextSeq 2000.