title: GSM5658008: BMDM_4sU_LPS_total_rep2; Mus musculus; RNA-Seq
library_construction_protocol: 4-thiouridine labelling and purification of nascent transcripts was performed according to the protocol published by Raedle at al. (doi: 10.3791/50195 ). In detail, RNA from Trizol-lysed and 4sU-labelled macrophages was isolated by chloroform-phenol extraction according to standard protocols. Shortly, 2 ml of chloroform was added to 10 ml of cell lysate, mixed vigorously and incubated for 2-3 mins at 20°C. After centrifugation (15 mins, 13,000xg at 4°C), the upper aqueous phase is transferred to a new 15-ml polypropylene tube. The RNA is precipitated with 0.5 volumes of RNA precipitation buffer (1.2M NaCl, 0.8 M sodium citrate) and 0.5 volumes of isopropanol and washed twice with 75% ethanol (centrifugation for 10 mins at 13,000xg and 4°C). The RNA was resolved in RNase-free water and the concentration measure using a Nanodrop1000 (ThermoFisher Scientific). For Thiol-specific biotinylation of the nascent (4sU-incorporated) RNA, 80 mg of total RNA were incubated with 1 mg/ml EZ-Link Biotin-HPDP (solubilized in DMF, Pierce, Cat.No: 21341) in biotinylation buffer (10 mM Tris pH=7.4, 1 mM EDTA) for 1.5 hrs. Subsequently, the total RNA was purified by chloroform-phenol extraction and precipitated with isopropanol in high salt conditions. The total RNA is diluted to 1 mg/ml and denatured (10 min 65°C). Streptavidin pulldown using streptavidin-coated magnetic beads (Miltenyi Biotec, Cat. No: 130-074-101) and mMACS columns (Miltenyi Biotec, Cat.No: 130-074-101) was performed to capture the fraction of nascent (biotinylated) RNAs. Shortly, columns are pre-equilibrated in washing buffer (100 mM Tris pH=7.4, 10 mM EDTA, 1M NaCl, 0.1% Tween-20), 100 ml streptavidin beads added to 100 ml 1 mg/ml biotinylated RNA and loaded to the column. Beads are washed three-times in pre-warmed (65°C) washing buffer and three-times in washing buffer at 20°C. RNA is eluted twice with 100 ml of 100 mM DTT into 400 ml of magnetic Agencourt RNA clean XP beads (Beckman Coulter, Cat.No: A63987). The nascent RNA is purified using Agencourt RNA clean XP beads as follows: 5 mins incubation at 20°C, 5 min incubation on the magnetic rack at 20°C, removal of the supernatant, 1x wash with 1 ml 70% ethanol, 5 min incubation on the magnetic rack at 20°C, removal of ethanol, air-dry Agencourt beads for approximately 10 mins at 20°C, elution in 30 ml RNase-free water. The concentration of the nascent RNA was quantified using the Qubit2.0 (ThermoFisher Scientific) and the RNA quality judged by Bioanalyzer (Bioanalyzer2100, Agilent Technologies) using the RNA 6000Nano Reagents (Agilent Technologies, Cat.No: 5067-1511). Depletion of rRNAs (Ribo-Zero Gold rRNA Removal Kit (Illumina)) and library preparation was performed starting with 400 ng of nascent RNA using the TruSeq Stranded Total RNA Library Prep Kit (Illumina, total RNA) or the TrueSeq Stranded mRNA Library Prep Kit (Illumina, PolyA-RNA). Amplified cDNA libraries were further purified using Agencourt RNAClean XP Beads (Beckman Coulter) and quality control of biotinylated RNA and cDNA libraries were performed using Agilent Bioanalyzer2100 with the RNA 6000Nano Reagents (Agilent Technologies, Cat.No: 5067-1511) or High Sensitivity DNA Reagents (Agilent Technologies, Cat.No: 5067-4626). RNAseq (total and PolyA)