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Sample GSM7781630 Query DataSets for GSM7781630
Status Public on Apr 24, 2024
Title iMGL_Scrambled_48h_1.25uM_rep1
Sample type RNA
 
Source name iPSC derived microglia
Organism Homo sapiens
Characteristics aso: Scrambled
treatment time (h): 48
dose (microm): 1.25
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 2.830910978
AFFX-BkGr-GC04_st 2.863475642
AFFX-BkGr-GC05_st 2.822228414
AFFX-BkGr-GC06_st 2.871043445
AFFX-BkGr-GC07_st 2.914236338
AFFX-BkGr-GC08_st 2.823208277
AFFX-BkGr-GC09_st 2.939813292
AFFX-BkGr-GC10_st 3.032353264
AFFX-BkGr-GC11_st 2.896625565
AFFX-BkGr-GC12_st 2.985928278
AFFX-BkGr-GC13_st 3.029217723
AFFX-BkGr-GC14_st 3.058383177
AFFX-BkGr-GC15_st 3.192445951
AFFX-BkGr-GC16_st 3.245656434
AFFX-BkGr-GC17_st 3.584930036
AFFX-BkGr-GC18_st 3.600121212
AFFX-BkGr-GC19_st 3.659309958
AFFX-BkGr-GC20_st 4.273951794
AFFX-BkGr-GC21_st 4.91604779
AFFX-BkGr-GC22_st 5.597678726

Total number of rows: 22593

Table truncated, full table size 673 Kbytes.




Supplementary file Size Download File type/resource
GSM7781630_108327.CEL.gz 414.1 Kb (ftp)(http) CEL

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