title: 4tU RNA-seq from wild-type and Rpb7-QHF cells
design: 4tU RNA-seq from wild-type and Rpb7-QHF cells
library_construction_protocol: Overnight cultures of three independent biological replicates of wild-type (WT) and Rpb7-QHF cells were subcultured to an OD600 of 0.2 and grown to an OD600 of ~1. Nascent RNA was labelled by adding 5 mM 4-thiouracil (Sigma) to the cell culture for 6 minutes. Cells were harvested by centrifugation, flash frozen in liquid N2 and kept at -80°C. S. pombe spike-ins were prepared by subculturing 10 ml of overnight wild-type cells into 500 ml of YES media and grown to an OD600 of ~1. Nascent RNA was labelled by adding 5 mM 4tU for 6 minutes. Cells were harvested and resuspended in PBS to a cell density of 15 OD/ml. 1 ml aliquots were prepared and cell pellets were flash frozen for later use. To have equally distributed spike-in RNA across strains and replicates, six S. pombe spike-in aliquots were resuspended in 6 ml of TRI reagent (ThermoFisher, AM9738) and used to resuspend three biological replicates of WT or Rpb7-QHF cells. RNA was chloroform extracted and isopropanol precipitated following TRI reagent instructions. Extracted RNA was DNaseI treated (NEB, M0303S) for 1 hour at 37°C, and purified by phenol-chloroform extraction and isopropanol precipitation. RNA quality (i.e. intact rRNA) was assessed on an agarose gel. To deplete the ribosomal RNA (rRNA), we used the Ribominus Transcriptome Isolation Kit (Yeast and Bacteria) (Invitrogen K1550-03), and purified the RiboMinus RNA following the ethanol precipitation protocol included in the kit. Complete rRNA depletion was confirmed by analysis of RiboMinus RNA on an agarose gel. To biotinylate the nascent 4tU-labelled RNA, we used 3 μg of RiboMinus rRNA-depleted RNA in the biotinylation reaction (0.2 mg/ml EZ-Link Biotin-HPDP in 10 mM HEPES pH 7.5, 1 mM EDTA). Excess biotin was removed by phenol:chloroform:isoamyl alcohol extraction (25:24:1) and ethanol precipitation. Biotinylated RNA was purified with Dynabeads™ MyOne™ Streptavidin C1 (Thermo), washed with high salt buffer (100 mM Tris-HCl pH 7.5, 10 mM EDTA, 1M NaCl, 0.1% Tween-20) and eluted twice with 5% 2-merchaptoethanol. Nascent RNA was ethanol-precipitated, washed and resuspended in DEPC-treated water. Purified nascent RNA samples were used to prepare strand-specific libraries using the NebNext Ultra II Directional RNA-seq kit. Following library amplification (13 cycles), library concentration and size distribution were assessed using the Agilent 2100 Bioanalyzer instrument (High Sensitivity DNA Assay).