library_construction_protocol: mRNA: To harvest, cells were washed off the 6-wells with 500 uL ice cold PBS and then centrifuged for 1 minute at 1000g in a tabletop centrifuge. PBS was removed, cells were resuspended in 1 mL TRIzol (Thermo Fisher Scientific, 15596018) and RNA extracted according to the manufacturer protocol. Subsequently RNA was further purified via the RNeasy Mini Kit (Qiagen, 74104), including DNase digestion (RNase-Free DNase Set, Qiagen, 79254). Nascent RNA (nRNA): Cells were fractionated and nascent RNA was extracted from chromatin as described in Reimer K, Neugebauer K, Curr Prot 2020 (PMID: 33440169). For Illumina short read sequencing, the protocol was followed until step 40 (including poly(A)+ depletion but excluding ribosomal RNA (rRNA) depletion) and samples were subsequently used for library preparation, that included rRNA depletion (see below). For PacBio long-read sequencing of nascent RNA, rRNA depletion was carried out following the complete protocol. mRNA-Illumina sequencing: Extracted RNA was used to prepare RNA-seq libraries using the KAPA mRNA HyperPrep Kit (Kapa Biosystems). The RNA-seq library was sequenced on the Illumina Novaseq X Plus platform generating ~25 milliion paired-end reads per sample. nRNA-Illumina sequencing:  Extracted RNA was used to prepare RNA-seq libraries using the KAPA RNA HyperPrep Kit with RiboErase (Kapa Biosystems). The RNA-seq library was sequenced on the Illumina Novaseq X Plus platform generating ~100 milliion paired-end reads per sample. mRNA-PacBio: The Iso-Seq libraries were prepared according to PacBio's Preparing Iso-Seq® Libraries using SMRTbell® Prep Kit 3.0 manual. 1.05x SMRTbell clean-up was done to size select 500bp and above fragments and one SMRT library was prepared and run on two SMRTcells. Primer annealing and polymerase binding reactions were prepared using the Binding Calculator from PacBio, based on sample volume, concentration, and insert size using default settings. The SMRTbell libraries were annealed to sequencing primers and then bound to polymerase using the Sequel® II Binding Kit 3.1 before being loaded onto the PacBio Sequel II sequencer. Sequencing was performed for 24 hours of movie time.