Dynabeads myone carboxylic acid

The single-cell RNA library was constructed according to our previously published method [13 (link)]. Briefly, the zona pellucida of oocytes was removed using Tyrode’s solution and digested in lysis buffer (1× PCR buffer II (without MgCl₂), 1.35 mM MgCl₂, 0.45% NP40, 4.5 mM DTT, 0.18 U/μL SUPERase-In, 0.36 U/μL RNase inhibitor, Thermo Fisher Scientific, Waltham, MA, USA). The supernatant was vortexed sufficiently and then centrifuged at 1000× g for 5 min at 4 °C. The DNA was isolated by Dynabeads Myone Carboxylic Acid (Thermo Fisher Scientific) for methylation-seq. Then, the UP1 primer (ATATGGATCCGGCGCGCCGTCGACTTTTTTTTTTTTTTTTTTTTTTTT) was added to the RNA lysate. The lysate was incubated at 70 °C for 90 s and immediately placed on ice. Then, the cDNA was synthesized by SuperScript III reverse transcriptase (Thermo Fisher Scientific). The extra primer was removed by ExoSAP-IT (Thermo Fisher Scientific). The polyA tail was added by the TdT. The UP2 primer (ATATCTCGAGGGCGCGCCGGATCCTTTTTTTTTTTTTTTTTTTTTTTT) was ligated to the cDNA by PCR. The cDNA was amplified with 24 cycles of PCR. The sequencing library was constructed by NEBNext Ultra DNA Library Prep Kit (NEB, Ipswich, MA, USA) according to the instruction. The library was sequenced using the Illumina platform. All chemicals were purchased from Sigma Aldrich, St. Louis, Mo, USA, unless mentioned otherwise.

The cryo-EM grids (200 mesh holey carbon R1/4 AU grid, Quantifoil Micro Tools) were glow-discharged to increase the hydrophilicity. After 30 min UV radiation in a laminar flow hood, the cells were seeded on the grids. Then, the prepared grids with the infected cells were subjected to plunge-freezing. For this, 4 µL blotting buffer (PBS, 10% glycerol, 0.5 mg/mL Dynabeads™ MyOne™ Carboxylic Acid) was added to the cryo-EM grids in a Vitrobot Mark IV (Thermo Fischer Scientific) with 90% humidity at 37 °C. The blot force and time were 10 and 10 s, respectively. The cryo-EM grids were stored in liquid nitrogen until use.

Cells (5 × 10⁵) were seeded on Quantifoil silicon oxide grids R 1/4 finder F1 Au grids, 200 mesh (Quantifoil Micro Tools GmbH, Jena, Germany). Cells were then cultured in RPMI medium (24 h, 37°C, 5% CO₂) and stained with MitoTracker Red FM (M22425; ThermoFisher Scientific) for 30 min at 37°C. Grids were vitrified by plunge-freezing using a Leica EM GP2 grid plunger (Leica Microsystems, Vienna, Austria) set to 37°C, 95% humidity and a blotting time of 7 s by the grid side opposite to the growing cells. Immediately before vitrification, 3 μl of Dynabeads MyOne Carboxylic Acid (1 μm; ThermoFisher Scientific) were added at a concentration of 0.5 mg/ml to each of the samples. Vitrified grids were mounted under liquid nitrogen in c-clip rings (ThermoFisher Scientific).

After the appropriate experiments, cells were lysed in ice-cold RIPA buffer (150 mM NaCl, 50 mM Tris-HCl pH 8.0, 1% TritonX-100, 0.1% sodium deoxycholate, 0.1% sodium dodecyl sulphate, 1 mM Na₃VO₄) supplemented with Complete Mini Proteases Inhibitor Cocktail (Roche, Basel, Switzerland). Cell lysates generally were mixed to micromagnetic beads conjugated to the appropriate antibody and left to react for 5 h at 4 °C with continuous rotation. Micromagnetic beads (“Dynabeads MyOne Carboxylic Acid”, Thermo Fisher Scientific, Waltham, MA, USA) covalently conjugated with the appropriate antibody were prepared following manufacturer’s instructions for “two step coating procedure”. Immunoprecipitated proteins were eluted from the antibody linked to the beads by denaturation in the same protein loading buffer used for Western blotting.
ECFP/SHC3 fusion proteins were immunoprecipitated using the “μMACS GFP Isolation Kit” (Miltenyi Biotec, Bergisch Gladbach, Germany) following manufacturer’s instructions.

THP-1 cells (American Type Culture Collection) were cultivated in RPMI 1640 medium (Sigma-Aldrich) supplemented with 10% fetal calf serum and 2% penicillin-streptomycin, at 37°C with 5% CO₂. Cells were plated in a 24-well plate over glass microslides at 500,000 cells per well and differentiated into macrophages with phorbol 12-myristate 13-acetate (2 μM, Sigma-Aldrich). At 24 hours, cells in suspension were removed and 700-nm M3P or nondegradable MPIO (Dynabeads MyOne Carboxylic Acid, Thermo Fisher Scientific) were added at a final iron content of 15 μg/ml. At 96 hours, THP-1 macrophages were processed for transmission electron microscopy (TEM).

The protein antigens (CMV, T-D and NC) were covalently linked to 1 μm paramagnetic polystyrene beads (Dynabeads MyOne Carboxylic acid, Thermofisher) using amine coupling. A magnet (Magrack 6, GE) was used to facilitate recovery of the beads between each following steps. To keep the beads in suspension all incubation steps were performed in 1.5 ml tubes on a shaker block at ≥900 RPM. In order to minimize bead losses when handling the beads, low retention tubes and pipette tips were used throughout the whole procedure. The beads were first washed twice in 25 mM MES buffer, pH 6, followed by incubation for 30 min at room temperature with 0.1 M N-hydroxysuccinimide (NHS) (Sigma-Aldrich) and 0.4 M 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (Fisher Scientific) in MES-buffer. After washing twice in MES-buffer, the antigen was added at a concentration of 1 mg/ml in MES-buffer, 67 μg protein/mg (n = 10⁹) beads, and incubated for 30 min at room temperature. After antigen coupling, still reactive carboxylic groups were blocked by incubating with 50 mM Tris pH 7.4 for 15 min.