35 μm cell strainer

Transfected cells were lifted and passed through a 35μm cell strainer (Falcon, 352235). Cells were gated to have a narrow range of FCS and SSC values. Autofluorescence was detected by the 405nm laser and 450/50 filter. TDP-43 reporter fluorescence was detected using the 488nm laser and 515/510 filter, and aggregation was quantified by comparing the height (FITC-H) to the width (FITC-W) of the fluorescence channel.

For flow and imaging cytometry cells were washed twice with 2.5mmol EDTA in PBS, detached with trypsin-EDTA solution (0.05%) (Thermo, Reinach, Switzerland) and put through a 35μm cell strainer (Falcon, Corning, USA) after resuspension. The fixable viability dye eFluor® 660 (e Bioscience, Hatfield, UK) was used for viability analysis in accordance with the manufacturer’s instructions. For cell analysis, BD FACS Canto II (BD) and Image Stream X Mark II (Merck, Millipore, Schaffhausen, Switzerland) were used. Doublets were excluded with the aid of a pulse geometry gate (FS-H x FS-A) in the flow cytometer and microscopically in the Image Stream analysis. Dead cells were excluded upon staining. For the flow cytometry analysis, the FSC-A voltage values of the first 500 cells in each experiment were analyzed and normalized to normoxia. For image stream analysis, cell size for every measured cell was determined. ANOVA and Bonferroni correction was used for comparison of the different conditions.

The tissue was received in ice-cold PBS and enzymatically digested to obtain the single-cell suspension. For this, tissue cut into smaller pieces was incubated with 500 μl 0.05% Trypsin/0.02% EDTA (Sigma, 59417-C) for 15 min at 37 °C with gentle swirling every 5 min. Gentle pipetting up and down was used to mechanically dissociate bigger pieces if any. 500 μl 10% FBS in PBS was added to cell suspension and the cells were pelleted at 500 × g for 5 min at 4 °C. Cells were washed two times with 1000 μL PBS, passed though the 35 μm cell strainer (Falcon, 352235), pelleted at 500 × g for 5 min at 4 °C and re-suspended in 100 μl 0.04% BSA in PBS. Ice-cold methanol (400 μL was added for fixation of the cells and the cells were stored at −80 °C. For the preparation of the library the cells were brought to +4 °C and pelleted at 1000 × g for 10 min at 4 °C. Cell pellet was re-suspended in 500 μL of rehydration buffer (1X DPBS (Gibco 14190144) containing 1.0% BSA (Sigma, B4287) and 0.5 U/μl RNAse Out (ThermoFisher Scientific, 10777019) followed by two washes with 500 μL of rehydration buffer. The rehydrated cell suspension was sorted to remove debris with BD FACS Aria Fusion instrument (BD Biosciences, San Jose, CA) equipped with 100 μm nozzle. After FACS cells were pelleted to obtain concentrated cell suspension with 700–1200 cells/μL.

All human fetuses were washed in cold PBS for 3–4 times, and then the thymus lobes were dissected in pre-cooled RPMI 1640 medium (Gibco, 11875093) containing 10% fetal bovine serum (FBS, Gibco, 10437-028). Cell suspension was prepared by gently smashing the thymus through a 70-μm cell strainer (Falcon, 352350). The suspension was treated with 1× red blood cell (RBC) lysis buffer (eBioscience, 00-4333-57) at 4°C for 5 min, and the remaining cell suspension was washed with PBS + 2% BSA. After filtering through a 35-μm cell strainer (Falcon, 352235), cells were stained with 7-AAD (BioLegend, 420403), and 7-AAD^– viable cells were sorted using a BD FACSAria III cell sorter and finally resuspended in PBS + 0.04% BSA at a concentration of 700–1,200 cells/μL.
Cell viability was measured by a trypan blue assay, and samples with more than 80% viability were used for library construction. Then scRNA-seq libraries were prepared using a Chromium Single Cell 3′ Reagent Kit (Version 2, 10× Genomics) (Zheng et al., 2017 (link)) following the manufacturer’s protocol. The cDNA libraries were sequenced on an Illumina HiSeq X Ten platform in the paired-end 150 bp mode.