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Sample GSM3717633

Query DataSets for GSM3717633

Status

Public on May 14, 2020

Title

CD21_1 iMG

Sample type

SRA

Source name

CD21 hiPSC-derived microglia

Organism

Homo sapiens

Characteristics

cell line background: CD0000021 (CD21)
cell type: human induced pluripotent stem cell (hiPSC) line
cell subtype: CD21 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

CD21_1_USR18003094L_HCCTMDMXX

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