title: GSM6733544: WT_SCoV2_4_6hpi_rep2; Homo sapiens; RNA-Seq
library_name: GSM6733544
library_construction_protocol: Cells were harvested in Trizol and RNA was extracted using the Direct-zol RNA microprep kit (Zymo Research) including DNase digest. From each sample, 2 μg of RNA were subjected to alkylation by incubating with 10 mM Iodoacetamide in PBS with 50% DMSO at 50°C for 15 min as previously described (Herzog, V.A., et al. Thiol-linked alkylation of RNA to assess expression dynamics. Nat Methods 14, 1198–1204 (2017). After adding an equal volume of H2O to the sample, the RNA was purified using the RNA Clean and Concentrator-5 kit (Zymo Research). Approximately 800 ng of alkylated RNA was depleted of ribosomal RNA using the NEBNext rRNA Depletion Kit v2 (NEB) according to the manufacturer's instructions. RNA was heat fragmented at 91°C for 3 min in 10 mM Tris-HCl pH 8.0, 5 mM MgCl2, 100 mM KCl, 0.02% Triton X-100 and 0.1 mg/mL BSA. RNA was dephosphorylated using 1.75 U FastAP Thermosensitive Alkaline Phosphatase for 10 min at 37°C , and end-repaired using 15 U T4 PNK for 20 min at 37°C. Both reactions were performed in the presence of 10 U murine RNase inhibitor. RNA was cleaned up using Dynabeads MyOne Silane (Thermo Fisher Scientific). For this, 10 μl beads per sample were washed twice with RLT buffer (Qiagen) and beads were added to the RNA in 3x sample volumes RLT buffer. To bind the RNA to the beads, 4.5x volumes isopropanol were added and samples were incubated 5 min at room temperature. Beads were washed twice with 70% EtOH and RNA was eluted in 8 μl H2O. cDNA libraries were constructed from the recovered RNA as described in the Methods section of the accompanying paper.