Rnasin plus ribonuclease inhibitor

Selected genes of interest were quantified using real-time reverse transcriptase quantitative polymerase chain reaction (qPCR). From cell culture, CD4⁺ T cells were harvested, and mRNA isolated using NucleoSpin RNA Mini Kit (MACHEREY-NAGEL, Cat# 740955.5) according to the instructions. The cDNA was transcribed using Promega M-MLV Reverse Transcriptase (Cat# M1701), RNasin Plus Ribonuclease Inhibitor (Cat# N2611), oligo(dT) 15 Primer (Cat# C1101), and PCR Nucleotide Mix (Cat# C1141). SYBR Green PCR Master Mix (Applied Biosystems, Cat# 4309155) was used for qPCR analysis, the primers used are detailed in Table S1.

DNA/RNA oligos were labeled with ³²P at the 5’ end using ATP-γ−³²P (PerkinElmer) and T4 PNK kinase (NEB) according to the NEB online protocol for radioactive labeling. Oligos after labeling were purified using a mini Quick Spin DNA Column (Roche) to remove unincorporated ATP-γ-³²P. TOP3B DNA cleavage assay was carried out in a 20 μl reaction containing 5 mM Tris-HCl (pH 7.5), 100 mM potassium glutamate, 0.02% Tween 20, 2 mM DTT, with addition of indicated amount of MnCl₂/MgCl₂/EDTA. Buffer for RNA cleavage assays contains 1X RNasin® Plus Ribonuclease Inhibitor (Promega). Final concentrations of labeled DNA/RNA oligo and TOP3B/TOP3B mutant were 6 nM and 18 nM. After incubation at 30 °C for 30 min or other indicated period of time, cleavage products (20 μl) were mixed with 20 μl 2X Formamide gel-loading buffer (10 mM EDTA, 0.025% bromophenol blue, 0.025% Xylene cyanol FF and 0.2% SDS dissolved in formamide), heat denatured at 95 °C for 3 min, and separated on a 18% acrylamide gel containing 7 M Urea. Gel was then dried and imaged with a GE Typhoon Phosphorimager.
To observe reversal of TOP3B cleavage product, 2.2 μl of 10X reversal buffer containing 5 mM Tris-HCl (pH 7.5), 5 M NaCl, and indicated metal/EDTA concentration was added to the 20 μl reaction sample and incubated for required time before being terminated by mixing with the 2X Formamide gel-loading buffer.

PBMC was thawed in phosphate-buffered saline (PBS) containing 2% bovine serum albumin (BSA) and stained with LIVE/DEAD™ Fixable Violet stain (ThermoFisher SCIENTIFIC, Waltham, MA, USA) for 30 min at 4 °C. Cells were washed and then stained with anti-horse antibodies or antigen at concentrations between 0.2–1 μg/mL. After washing again, cells were resuspended in 2 mL PBS (2% BSA) and sorted as IgG+ and antigen single or double positive, at one cell per well, into PCR plates containing Rnasin^® Plus Ribonuclease Inhibitor (Promega, Madison, WI, USA) and IGEPAL CA-630. At completion, PCR plates were flash frozen in liquid nitrogen and stored at −80 °C.

Culture aliquots (1 ml) were collected at OD₆₀₀ 0.5, centrifuged for 3 min at 8000 rpm and the pellet was washed once with 1 mL 1X TE buffer (10 mM Tris HCl pH 8.0, 1 mM EDTA). The pellet was resuspended in 100 μL lysostaphin buffer composed of 200 μg mL⁻¹ lysostaphin, 200 μg mL⁻¹ DNase I, 40 U RNasin Plus Ribonuclease inhibitor (Promega) in 1X TE and placed at 37 °C for 10 min. RNA was then extracted using RNeasy Plus Mini Kit (Qiagen) following the manufacturer’s instructions and adding two modifications: cell homogenization was performed with QIAshredder (Qiagen) columns after adding RTL Plus lysis buffer and DNase I treatment using RNase-Free DNase Set (Qiagen) was applied after first column wash.