aso: Untreated treatment time (h): 48 dose (microm): 0
Treatment protocol
ASOs were added directly into the medium of the microglia on DIV7. ASOs were incubated for 24 and 48hours. Medium was removed and cells were lysed in 150uL RLT buffer (Qiagen, 74182).
Growth protocol
The iPSC lines used in the cellular assays were obtained from the EBiSC consortium (https://ebisc.org) (Suppl Table 2). The human iPSC lines BIONi010-C (parental; CVCL_1E68) was used to evaluate the off target effects of the ASOs in vitro. Human iPSC-derived microglia (iMGL) were differentiated using a method described by Cowley lab (Wilgenburg, B. van, Browne, C., Vowles, J. & Cowley, S. A. Efficient, Long Term Production of Monocyte-Derived Macrophages from Human Pluripotent Stem Cells under Partly-Defined and Fully-Defined Conditions. PLoS One 8, e71098 (2013)/Haenseler, W. et al. A Highly Efficient Human Pluripotent Stem Cell Microglia Model Displays a Neuronal-Co-culture-Specific Expression Profile and Inflammatory Response. Stem Cell Reports 8, 1727–1742 (2017)). Briefly, iPSCs were dissociated with TrypLE select (ThermoFisher, 12563011) into single cell suspension and embryoid bodies (EBs) were produced in AggreWell800 (Stem Cell Technologies, 34811) in mTeSR medium (Stemcell Technologgies, 85850) supplemented with 50 ng/ml BMP-4 (Invitrogen, PHC9534), 50 ng/ml VEGF (PeproTech, 100-20) and 20 ng/ml SCF (Miltenyi, 130-096-693). The medium was refreshed daily by 75% for 3 consecutive days. Next, EBs were transferred to factory plates to create precursor factories by placing 10-20 EBs per well in X-VIVO 15 medium (Lonza, BE02-060F) containing 100 U/ml Penicillin and 100 µg/ml Streptomycin (ThermoFisher, 15140-122), 2 mM Glutamax supplement, 0.05 mM 2-mercaptoethanol (ThermoFisher, 31350-010), 0.1 µg/ml M-CSF (ThermoFisher, PHC9501) and 0.025 µg/ml IL-3 (ThermoFisher, PHC0031). The factories were cultured for 6-8 weeks (with weekly 50% medium change) before the cells were harvested for final differentiation into microglia. Microglia precursors were harvested for no more than once a week. At harvest, microglia precursors were plated at a density of 15,000 cells/well in non-coated 96-well µclear plates (Greiner, 655090) and cultured in advanced DMEM/F12 medium (ThermoFisher, 12634-010) containing 100 U/ml Penicillin and 100 µg/ml Streptomycin, 2 mM Glutamax supplement, 0.05 mM 2-mercaptoethanol, 10 ng/ml GM-CSF (ThermoFisher, PHC2015) and 100 ng/ml IL-34 (Peprotech, 200-34) for maximally 14 days (with 50 % medium change every 2-3 days).
Extracted molecule
total RNA
Extraction protocol
Total cellular RNA was isolated using the RNeasy96 kit (Qiagen, 74182) according to manufacturer’s protocol. Briefly, cells were lysed with RLT buffer at RT and an equal volume of 70% (v/v) ethanol was added. The mixture was pipetted 5 times and transferred to columns in which the RNA was bound to the filter by a vacuum system (Qiagen). The RNA was washed sequentially with the RW1 and RPE buffers provided in the kit. After the final RPE washing step, a centrifugation was applied to make sure any residual buffer was eliminated (6 min at 5,600 x g at room temperature (RT). The RNA was then eluted with RNase-free water by centrifugation at 5,600 x g at RT for 4 min. The RNA concentration was determined by spectroscopy using a Nanodrop (ThermoFisher, ND-8000).
Label
biotin
Label protocol
Amplification and labelling of total RNA were performed using the GeneChip® PICO Reagent Kit following the manufacturer’s protocol (ThermoFisher 2016, P/N 902790).
Hybridization protocol
Biotin-labeled target samples were hybridized to the Clariom™ GO Screen containing probes for 19,409 protein-coding genes.
Scan protocol
Target hybridization was processed on the GeneTitan® Instrument according to manufacturer’s instructions provided for Expression Array Plates (P/N 952361). Images were analyzed using the GeneChip® Command Console Software (GCC) (ThermoFisher).
Description
Off-target analysis of APOE and TREM2 ASOs in iPSC derived microglia
Data processing
Microarray data were processed using the statistical computing R-program package limma (Version 3.42.0) and Bioconductor tools. The gene expression values were normalized using Robust Multi-array Average (RMA). Individual probes were grouped into gene-specific probe sets based on Entrez Gene using the metadata package goscreenhuhsentrezg (version 25.0.0)
