title: GSM8673095: long-read, nascent RNA, JTE, 1h, biol rep 1; Homo sapiens; RNA-Seq
library_construction_protocol: Cell fractionation, nascent RNA isolation, polyA-RNA and rRNA depletion, and library preparation steps were performed following the protocol described by Reimer K, Neugebauer K, Curr Prot 2020 (PMID: 33440169). Following cell fractionation, RNA was isolated from the chromatin after Trizol/chloroform extraction using RNAeasy Mini Kit (including on-column treatment with RNAse-free DNAse). Then poly(A)+ RNA was depleted using DynaBeads mRNA Direct Micro Purification kit, followed by rRNA depletion using RiboMinus Eukaryote Kit v2. and ligation of a 3'end adapter to each nascent RNA. Libraries for PacBio sequencing were prepared following the protocol described by Reimer K, Neugebauer K, Curr Prot 2020 (PMID: 33440169). After poly(A)+ RNA and rRNA depletion steps, a 3'end DNA adapter with UMI was ligated to each nascent RNA. The PacBio HiFi library for SMARTER/Advantage2 PCR amplified cDNA size selected for fragments longer than 500nt was prepared using SMRTbell Express Template Prep Kit 2.0 from PacBio following the manufacturer's instructions.