Experion electrophoresis system

An RNeasy mini kit was used for RNA extraction from transfected HEK293T cells (Qiagen, Hilden, Germany) at different time-points. For the microarray experiment, RNA was extracted 12 h post-transfection. For the gene analysis by real time rtPCR, RNA was extracted at either 3, 6, 12, 15, 18, 20, or 24 h post-transfection. For the microarray experiment, the integrity of the resulting RNA was checked on an automated Experion electrophoresis system (Biorad, Hercules, CA, USA).

For bulk sequencing, total RNA was isolated from either sorted microglia or nuclei using the RNeasy Plus Micro Kit (Qiagen,74034). RNA quantity and quality were analyzed using an Experion electrophoresis system (Bio‐Rad Laboratories, Hercules, CA, USA). Sequencing libraries were prepared with the Quant Seq 3' mRNA‐Seq Library Prep Kit FWD (Lexogen, Vienna, Austria).
The single‐cell barcoded libraries were constructed using Single Cell 3' Reagent Kits v2 (10× Genomics). In brief, after sorting, single cells were partitioned into nanoliter‐scale Gel Bead‐in‐emulsions (GEMs) in the chromium controller. GEMs were then incubated in a thermal cycler to generate barcoded cDNA. After amplification, cDNAs were further processed for sequencing by ligation of adapters and individual sample indices. The libraries were sequenced on a NextSeq platform.

RNA extraction and library preparation were performed as described in Halstead et al. (2021) (link). In brief, frozen tissues were mashed using a pestle in a mortar filled with liquid nitrogen. Next Trizol reagent (Invitrogen, Carlsbad, CA, United States) was added to extract total RNA using the Direct-zol RNA Mini Prep Plus kit (Zymo Research, Irvine, CA, United States). The integrity and quality of the extracted RNA was checked using an Experion electrophoresis system (Bio-Rad, Hercules, CA, United States) and samples passing quality control were used for library preparation. First, 50 ng of total RNA in a volume of 9 μl was mixed with 1 μl 10 μM VNP primer and 1 μl 10 mM dNTPs, then incubated 5 min at 65°C. The resulting products were used for strand-switching and reverse transcription reactions (Halstead et al., 2021 (link)). Then barcodes were ligated to the cDNA products generated from the last step using the Oxford Nanopore PCR barcoding expansion 1-96 kit (cat. no. EXP-PBC096), which were further ligated with adapters from the SQK-DCS109 kit following the manufacturer’s guidelines. Products were loaded onto a PromethION flow cell (vR9.4.1) for sequencing.