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Sample GSM7906783 Query DataSets for GSM7906783
Status Public on Jan 10, 2024
Title PIMP_1
Sample type SRA
 
Source name cells
Organism Homo sapiens
Characteristics tissue: cells
cell type: human primitive macrophage precursors
Treatment protocol n/a
Growth protocol Human iPSCs-derived microglia (hiMG) were derived from human induced pluripotent stem cells (iPSCs). Human iPSCs were cultured in mTeSR1 (Stem Cell Technologies) on hESC-qualified Matrigel-coated plates (Corning). iPSCs were routinely passaged using accutase cell detachment solution (Stem Cell Technologies). Using an adapted protocol with minor modifications, microglia were differentiated from iPSCs via embryoid bodies (EBs) and primitive macrophage precursors (PMPs)86,87. iPSCs were dissociated to single cells with accutase cell detachment solution and plated at 10,000 cells per well in 96-well ultra-low attachment plates (Corning) in 100 μL of microglia embryoid body induction media (MEBIM) supplemented with the ROCK inhibitor StemMACS Y27632 (Miltenyi Biotec) prior to centrifugation at 300 x g for 3 min at room temperature (RT). Plated cells were then stored in a 5% CO2 incubator set to 37°C. EBs were cultured for 4 days with a half medium (i.e., 50 μL) exchange with MEBIM occurring after 2 days. When performing the half media change, small molecule concentrations were doubled to account for the media already present in the wells. After 4 days, 16 EBs were plated per well of tissue-culture treated 6-well plates in 3 mL of microglia differentiation medium A (MDM-A). From this point forward, 2 mL medium was exchanged every 5-6 days. When performing the two-thirds media change, small molecule concentrations were doubled to account for the media already present in the wells. After 14 days, PMPs were harvested from suspension during a media exchange. Harvested media was first filtered through a 40 μm nylon membrane then centrifuged at 500 x g for 5 min at RT. Pelleted cells were resuspended in an appropriate volume of microglia differentiation medium B (MDM-B) and a cell count performed using trypan blue (Thermo Fisher Scientific) exclusion. Cells were plated at a density of 180,000 – 200,000 cells/cm2 in 6-well plates in 1.5 mL of MDM-B. PMPs adhered to the uncoated tissue culture-treated plastic within 24 hrs. The maturation of the PMPs into microglia occurred over 14+ days, with half the medium exchanged every 3 days. When performing the half media change, small molecule concentrations were doubled to account for the media already present in the wells. All cytokines and growth factors were obtained from Peprotech. After a minimum of 14 days of maturation, microglia-like cells were incubated in 2 mL of accutase cell detachment solution for 20-30 min in a 5% CO2 incubator set to 37°C. After 20-30 min, any remaining attached cells were detached with gentle pipetting. Microglia-like cells were centrifuged at 500 x g for 5 min at RT. Pelleted cells were resuspended in an appropriate volume of MDM-B and a cell count performed using trypan blue exclusion. Cells were allowed to rest for 48 hrs prior to experiments being performed.
Extracted molecule polyA RNA
Extraction protocol Qiagen RNeasy Kit
standard Illumina protocol
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NovaSeq 6000
 
Data processing Quality checks were performed using FastQC (v0.12.1), summarised with MultiQC (v1.14). Reads were then subsampled. Alignment to the human reference genome (build GRCh38.p13) was performed using STAR (v2.7.10a) with default parameters. The counts of the aligned reads corresponding to exons were obtained using featureCounts program against the exon annotations obtained from the ensembl database (annotation file Homo_sapiens.GRCh38.101.gtf). MA plots, JSIs and PCA were performed as further quality checks.
The resulting expression values were normalised using quantile normalisation [Bolstad2003] and noise levels were assessed using noisyR count matrix approach [Moutsopoulos2021].
Differential expression analysis was performed using edgeR (v3.34.1) [Robinson2010] and DESeq2 1.32.0 [Love2014].
The visualisation, and community accessible overview were complied using bulkAnalyseR [Moutsopoulos2023].
Assembly: hg38 (GRCh38.p13)
Supplementary files format and content: count matrix containing raw gene expression
Supplementary files format and content: count matrix containing normalised and denoised gene expression
 
Submission date Nov 17, 2023
Last update date Jan 10, 2024
Contact name Irina Mohorianu
E-mail(s) data-submissions@stemcells.cam.ac.uk
Organization name University of Cambridge
Department Wellcome-MRC Cambridge Stem Cell Institute
Street address Puddicombe Way
City Cambridge
ZIP/Postal code CB2 0AW
Country United Kingdom
 
Platform ID GPL24676
Series (2)
GSE248170 Mitochondrial complex I activity in microglia sustains neuroinflammation [bulk]
GSE248175 Mitochondrial complex I activity in microglia sustains neuroinflammation
Relations
BioSample SAMN38302041
SRA SRX22560558

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA

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