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Sample GSM7781569 Query DataSets for GSM7781569
Status Public on Apr 24, 2024
Title iMGL_Untreated_24h_rep3
Sample type RNA
 
Source name iPSC derived microglia
Organism Homo sapiens
Characteristics aso: Untreated
treatment time (h): 24
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)
 
Submission date Sep 14, 2023
Last update date Apr 24, 2024
Contact name Lina Vandermeulen
E-mail(s) LVanderm@its.jnj.com
Organization name Johnson & Johnson
Department Neuroscience
Street address Turnhoutseweg 30
City Beerse
ZIP/Postal code 2340
Country Belgium
 
Platform ID GPL31262
Series (1)
GSE243243 Off target expression data from iPSC derived microglia treated with APOE/TREM2 ASOs for 24h/48h. The iPSC cells are from a wild type donor (BIONi10C).

Data table header descriptions
ID_REF
VALUE RMA

Data table
ID_REF VALUE
AFFX-BkGr-GC03_st 3.113441898
AFFX-BkGr-GC04_st 2.921416528
AFFX-BkGr-GC05_st 2.88223412
AFFX-BkGr-GC06_st 2.972559239
AFFX-BkGr-GC07_st 3.024729205
AFFX-BkGr-GC08_st 2.984281296
AFFX-BkGr-GC09_st 3.050510122
AFFX-BkGr-GC10_st 3.059714042
AFFX-BkGr-GC11_st 2.958595811
AFFX-BkGr-GC12_st 3.110880125
AFFX-BkGr-GC13_st 3.108884578
AFFX-BkGr-GC14_st 3.128692384
AFFX-BkGr-GC15_st 3.208124861
AFFX-BkGr-GC16_st 3.383523428
AFFX-BkGr-GC17_st 3.81521076
AFFX-BkGr-GC18_st 3.870891194
AFFX-BkGr-GC19_st 3.650205814
AFFX-BkGr-GC20_st 4.22333063
AFFX-BkGr-GC21_st 4.985383927
AFFX-BkGr-GC22_st 5.638765942

Total number of rows: 22593

Table truncated, full table size 673 Kbytes.




Supplementary file Size Download File type/resource
GSM7781569_108277.CEL.gz 400.0 Kb (ftp)(http) CEL

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