|
| Status |
Public on Sep 15, 2021 |
| Title |
iPS microglia Hyb87_rep 2 |
| Sample type |
SRA |
| |
|
| Source name |
iPS microglia treated with Hyb87
|
| Organism |
Homo sapiens |
| Characteristics |
treatment: Hyb87
sample type: iPS microglia
|
| Treatment protocol |
iMGs were collected on day 39 and seeded on a 24-well non-tissue culture treated plate coated with 10 μg/mL Hyb87 or control mouse IgG
|
| Growth protocol |
Human iPSCs were cultured on laminin-coated plates in StemFit containing penicillin and streptomycin. The cells were passaged every 6 to 7 days using 0.5 mM EDTA, seeded at the density of 2 × 104 cells/well on a 6-well plate, and maintained in the presence of 10 μM Y-27632. The culture media was replaced with StemFit containing penicillin and streptomycin in the absence of Y-27632 24 h after passaging. iPSC-derived hematopoietic progenitors (iHPCs) were generated using defined conditions with several modifications to previously published protocols. Briefly, 0.5 to 1 × 105 cells were plated per well in a tissue culture-treated 6-well plate (day 0). The cells were cultured in 2 mL StemFit containing Y-27632 for 24 h under normoxic conditions. Day 1: Media was changed to basal medium supplemented with 50 ng/mL FGF2, 50 ng/mL BMP4, 12.5 ng/mL activin-A, 10 μM Y-27632, and 2 mM LiCl. The cells were then placed under hypoxic cell culture conditions of 5% O2 and 5% CO2 for 2 days. Day 3: The cells were further maintained in basal media supplemented with 50 ng/mL each of FGF2 and VEGF under hypoxic conditions. Day 5: Media was changed to basal media containing 50 ng/mL FGF2, 50 ng/mL VEGF, 50 ng/mL TPO, 10 ng/mL SCF, 50 ng/mL IL-6, and 10 ng/mL IL-3. The cells were placed under normoxic conditions. Day 7 and day 9: Medium was changed to that used on day 5. iMGs were differentiated from iPSCs via embryoid bodies and HPCs using defined conditions with several modifications to a previously published protocol. Day 11: iHPCs was washed using iMG basal differentiation medium. After centrifugation, the iHPCs were gently suspended in iMG complete differentiation medium containing 25 ng/mL M-CSF, 100 ng/mL IL-34, and 50 ng/mL TGF-β and seeded at the density of 2 × 105 cells per well on 6-well plates. One milliliter iMG complete differentiation medium was supplemented after every 2 days. Day 23 and day 37: The cells were collected as iMGs and seeded in a 1:1 mixture of conditioned medium and iMG complete differentiation medium [34]. Cells were supplemented with 1 mL iMG complete differentiation medium after every two days.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Extraction of total RNA was performed according to the manufacturer's instructions (RNeasy, QIAGEN)
10 ng of total RNA was reverse-transcribed using the SuperScript VILO cDNA Synthesis Kit (Thermo Fisher Scientific) followed by library generation using the Ion AmpliSeq Transcriptome Human Gene Expression Kit. Libraries were diluted to 45 pM and pooled equally, with 9-10 individual samples per pool.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Ion Torrent Proton |
| |
|
| Data processing |
Base calling was performed using software provided with the Ion Torrent Proton sequencer (Torrent suite ver.5.10.1).
Sequenced reads were trimmed for adaptor sequence and masked for low-complexity or low-quality sequence, then mapped to hg19_AmpliSeq_Transcriptome_21K_v1 using software provided with the Ion Torrent Proton sequencer (AmpliSeqRNA v5.10.1.2).
Count data were calculated using software provided with the Ion Torrent Proton sequencer (AmpliSeqRNA v5.10.1.2).
Genome_build: hg19
Supplementary_files_format_and_content: Tab-delimited text files include gene symbol and gene expression (read count) for each sample.
|
| |
|
| Submission date |
Oct 09, 2020 |
| Last update date |
Sep 17, 2021 |
| Contact name |
Yuumi Okuzono |
| E-mail(s) |
[email protected]
|
| Phone |
810466322618
|
| Organization name |
Takeda Pharmaceutical Company Limited
|
| Street address |
26-Muraoka-higashi 2-chome
|
| City |
FUJISAWA |
| State/province |
KANAGAWA |
| ZIP/Postal code |
251-8555 |
| Country |
Japan |
| |
|
| Platform ID |
GPL17303 |
| Series (1) |
| GSE159333 |
Expression data of iPS microglia treated with TREM2 agonist antibody |
|
| Relations |
| BioSample |
SAMN16408241 |
| SRA |
SRX9272949 |
