title: GSM8688931: HeLa_Ctrl_5K_rPRO-seq_R1; Homo sapiens; OTHER
library_name: GSM8688931
library_construction_protocol: For rPRO-seq experiments were conducted following established protocols (see detailed protocol in supplementary file 1). Initially, a mixture of HeLa or mESC nuclei and Drosophila S2 cell nuclei as spike-ins was prepared. Nuclear run-on assays were executed by incubating nuclei at 30°C for 3 minutes with 25 μM Biotin-11-ATP/UTP/CTP/GTP (PerkinElmer). Subsequently, total RNA was extracted using Direct-zol RNA Miniprep and fragmented with 0.2 M NaOH for 10 minutes on ice, followed by purification of biotinylated nascent RNAs using streptavidin beads M-280 (Invitrogen). The 3' RNA adaptor ligation process employs pre-adenylated single-stranded DNAs (P-3' App-DNA), and T4 RNA ligase 2, truncated KQ, is utilized, incubated at 25°C for 1 hour. Additionally, on-bead 5' RNA end repair involved using RppH (NEB) to remove the 5' cap and repair the triphosphate, followed by PNK-mediated repair of the 5' hydroxyl. The hybridization of DBO involves incorporating the dimer blocking oligonucleotide (DBO) into the RNA within the DBO mix. This is followed by a thermocycler program and subsequent combination with the 5' adaptor ligation mixes in the RNA DBO mix, with a 1-hour incubation at 25°C. After adapter ligation, SuperScript III reverse transcription was employed. PCR amplification generated 140–350 bp libraries, which were size-selected using AMpure XP beads and sequenced using single-read runs on a NovaSeq X.