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Sample GSM3717627 Query DataSets for GSM3717627
Status Public on May 14, 2020
Title CD11_2 iMG
Sample type SRA
 
Source name CD11 hiPSC-derived microglia
Organism Homo sapiens
Characteristics cell line background: CD0000011 (CD11)
cell type: human induced pluripotent stem cell (hiPSC) line
cell subtype: CD11 hiPSC-derived microglia day 12 after plating PMPs
Growth protocol CD0000011 (CD11) and CD0000021 (CD21) hiPSC lines were used to generated iMG. We followed the Brownjohn et al method. These cell lines were cultured in mTeSR complete medium (85850, STEMCELL) with 2 ml daily media change. At least two days after passaging, iPSCs were passaged to single cells and plated at 10,000 cells per well in 96-well round bottom ultra-low attachment plates (Corning) in 100 ul EB media and plates were centrifuged at 300g for 3 min. EB media was prepared by adding to the complete mTeSR media BMP-4 (Thermofisher), SCF (Thermofisher), VEGF-121 (Peprotech) and ROCK inhibitor (Tocris). Cells were cultured for four days, with half media change after two days. On day four, 16 EBs were plated on a 6-well plate and cultured in 3 ml hematopoietic media with 2 ml media exchanges every five days. Hematopoietic media was prepared by adding to the X-VIVO 15 (04-744Q, Lonza) GlutaMax (Thermofisher), Pen/strep (Thermofisher), β-mercaptoethanol (Sigma), M-CSF (Thermofisher) and IL-3 (Thermofisher). After five days culturing EBs in hematopoietic media, PMPs started to appear in the suspension and were produced continuously in suspension for 34 days. After 10 days of culturing EBs, PMPs could be harvested from suspension and plated in RPMI 1640 (Thermofisher) at 180,000 cells/cm2 in 6- or 12-well plates. After one hour, PMPs adhered to surface and media was changed to complete microglia media. Full media changes were done every 2 days. Complete microglia media was prepared with RPMI 1640 by adding FBS (Thermofisher), Glutamax, Pen/strep, IL-24 (PeptroTech) and GM-CSF (PeproTech). Final differentiation of PMPs into microglia occurred over 6-10 days and cells could be kept for at least one month in this format.
Extracted molecule total RNA
Extraction protocol Total RNA from each stem cell line after PBS wash. Cells were lysed in RLT plus buffer, and total RNA extracted with the RNeasy mini kit (Qiagen). cDNA was reverse transcribed from RNA with a high-capacity cDNA reverse transcription kit (Applied Biosystems).
Total RNA was sent for sequencing to Novogene (en.novogene.com). Sequencing libraries were prepared using the NEB Nextera kit with customized adapters.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NovaSeq 6000
 
Description CD11_2_USR18003088L_HFCGHDMXX
Data processing Read trimming with Trimmomatic v0.36 argurments(PE IILUMINACLIP:$adapters:2:30:10 LEADING:30 TRAILING:30 SLIDINGWINDOW:4:15 MINLEN:20)
Read mapping with STAR 2.6.1d arguments(--outSAMtype BAM SortedByCoordinate --bamRemoveDuplicatesType UniqueIdentical --outFilterIntronMotifs RemoveNoncanonical --outFilterMismatchNmax 2 --outFilterScoreMinOverLread 0.30 --outFilterMatchNminOverLread 0.30 --alignSoftClipAtReferenceEnds No)
Counts with featureCounts 1.6.3 arguments(-p -B -C -D 2000 -t gene)
Genome_build: GRCh38 Release-94
Supplementary_files_format_and_content: Raw feature count expression matrix for all samples submitted
 
Submission date Apr 11, 2019
Last update date May 14, 2020
Contact name Robert Raymond Butler
Organization name Stanford University School of Medicine
Department Neurology & Neurological Sciences
Lab Longo
Street address 3172 Porter Dr., MC 5475
City Stanford
State/province CA
ZIP/Postal code 94304
Country USA
 
Platform ID GPL24676
Series (1)
GSE129630 Human iPSC-derived microglia are genetically relevant to Alzheimer’s disease
Relations
BioSample SAMN11394787
SRA SRX5667714

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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