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| Status |
Public on Feb 11, 2022 |
| Title |
Guide 1 rep1 |
| Sample type |
SRA |
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| Source name |
hiPSC WTC_Guide 1
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| Organism |
Homo sapiens |
| Characteristics |
cell line background: WTC #11
source cell type: human pluripotent stem cell (hiPSC) line
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| Treatment protocol |
EPS programming: WTC colonies reached 60%–70% of confluence. 12 hr after seeding, the conventional WTC medium (TeSR) was replaced with the LCDM medium as described in derivation of pluripotent stem cells with in vivo embryonic and extraembryonic potency 52: 240 mL DMEM/F12 (Thermo Fisher Scientific, 11330-032),240 mL Neurobasal (Thermo Fisher Scientific, 21103-049), 2.5 mL N2 supplement (Thermo Fisher Scientific, 17502-048), 5 mL B27 supplement (Thermo Fisher Scientific, 12587-010), 1% GlutaMAX (Thermo Fisher Scientific, 35050-061), 1% nonessential amino acids (Thermo Fisher Sci- entific, 11140-050), 0.1 mM b-mercaptoethanol (Thermo Fisher Scientific, 21985-023), penicillin-streptomycin (Thermo Fisher Scientific, 15140-122), and 5% knockout serum replacement (KSR, Thermo Fisher Scientific, A3181502, optional) and the following factors : 10 ng/ml hLIF, 1 mM CHIR 9902, 2 mM (S)-(+)-Dimethindene maleate (DiM), 2 mM Minocycline hydrochloride (MiH), 0.5-1 mM IWR-1-endo, and 2mM Y-27632. The N2B27-LCDM medium was changed daily. Dome-shaped colonies gradually emerged during this period. Then, 3-6 days later, 0.05% Trypsin-EDTA was used to trypsinize the cells for 3 min at 370 C in the incubator. LCDM base medium (without factors) was used to stop the trypsinization. The cells were washed off the surface of dish by pipetting the medium slowly up and down: they were collected in an appropriately sized tube and centrifuged at 1, 200 at room temperature for 3 min. The cells were re-suspended in an appropriate volume of N2B27-LCDM medium (according to the cell lines and growth ratio) and seeded into the plate with MEF feeders. For one six-well plate, approximately 50,000-100,000 cells per well were seeded. The split ratio was usually from 1:3 to 1:10. Then, the cells were incubated under 20% O2 and 5% CO2 at 370C. . Trophoblast differentiation: EPS Cells were incubated with 0.05% Trypsin-EDTA for 3 min at 370 C. TX base medium (DMEM/F12 w/o HEPES or L-Glu (21331-020), 64mg/ml ascorbic acid phosphate Mg, 14ug/l sodium selenite, 19.4mg/l insulin, 10.7 mg/l holo-transferrin, 543mg/l NaHCO3, 1% Pen Strep and, 2mM Glutamax) (without factors) was applied to stop the trypsinization. 50k-100k cells were plated on 35mm matrigel (1:30) coated plate. TX Medium was prepared without growth factors and stored at 40C. TX factors (25ng/ml FGF4, 2ng/ml TGF-beta1and 1 mg /mL Heparin) were added to TX media prior to use. TX media was changed every other day. Cells were passaged when sub-confluent.
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| Growth protocol |
The hiPSC line WTC #11, previously derived in the Conklin laboratory, were cultured on Matrigel (1:30) growth factor-reduced basement membrane matrix (Corning) in mTeSR media (StemCell Technologies). Cells were passaged using versene once reaching 70% confluency and plated at 1:6 density. For EPS conditions, cells were grown as previously described. Briefly, WTC cells were reprogrammed in base medium containing 100 mL DMEM/F12, 100 mL Neurobasal, 1 mL N2 supplement, 2 mL B27 supplement, 1% GlutaMAX , 1% NEAA, 0.1 mM β-mercaptoethanol, penicillin-streptomycin and 5% KSR, and freshly supplemented with 10 ng/ml hLIF, GSK3i (1 μM), ROCKi (2 μM ), (S)-(+)-Dimethindene maleate (2 μM; Tocris), Minocycline hydrochloride (2 μM; Santa Cruz Biotechnology) and IWR-endo-1 (0.5-1 μM; Selleckchem). Cells were adapted to EPS conditions for at least 3 passages before analysis. EPS cells were pushed toward differentiation using TX media: TX medium formulation was DMEM/F12 without HEPES and L-glutamine (Life Technologies), 64 mg/l l-ascorbic acid-2-phosphate magnesium, 14 mg/l sodium selenite, 19.4 mg/l insulin, 543 mg/l NaHCO3, 10.7 mg/l holo-transferrin (all Sigma-Aldrich), 25 ng/ml human recombinant FGF4 (Reliatech), 2 ng/ml human recombinant TGF-ß1 (PeproTech), 1 mg/ml heparin (Sigma-Aldrich), 2 mM L-glutamine, 1% penicillin, and streptomycin (all PAN-biotech). Medium was prepared without growth factors (TX-growth factors) and stored at 40 C. To prepare complete TX, the growth factors: FGF4, heparin, and TGF-b1 were added prior to use. Medium was changed every other day. All cells were cultured at 370 C in 5% CO2. For Elf1 iCas9 conditions, cells were treated as described before but toggled to ‘primed’ cell state for efficient gRNA transfection. Briefly, Elf1 iCas9 were cultured on Matrigel (1:30) growth factor-reduced basement membrane matrix (Corning) in mTeSR media (StemCell Technologies). Cells were passaged using versene once reaching 70% confluency and plated at 1:6 density.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using Trizol (Life Technologies) according to manufacturer’s instructions. RNA samples were treated with Turbo DNase (Thermo Fisher Scientific) and quantified using Nanodrop ND-1000. Reverse transcription was performed using iScript cDNA Synthesis Kit (Bio-Rad). 10 ng of cDNA was used to perform qRT-PCR using SYBR Green (Applied Biosystems) or TaqMan (Applied Biosystems) on a 7300 real time PCR system (Applied Biosystems). The PCR conditions were set up as the following: stage 1 50°C for 2mins, stage 2 as 95°C for 10mis, 95°C for 15sec, 60°C for 1min (40 Cycles). ß-actin was used as an endogenous control. The primer sequences used in this work are shown in Supplementary Table S5.
RNA libraries were prepared for sequencing using standard Illumina protocols
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
V300012058_L*_HK500HUMotoEAAARBAPEI-568
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| Data processing |
RNA-seq reads in fastq files were aligned to hg19 using Hisat (version 2.0.5).
Gene-level read counts were quantified using htseq-count 62 using Ensembl GRCh37 gene annotations.
FeatureCounts was used to do gene-level quantification.
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited file of gene-level read counts for each sample.
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| Submission date |
Jul 23, 2020 |
| Last update date |
Feb 11, 2022 |
| Contact name |
Hannele Ruohola-Baker |
| E-mail(s) |
hannele@u.washington.edu
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| Organization name |
Institute for Stem Cell and Regenerative Medicine
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| Street address |
850 Republican Street
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| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98109 |
| Country |
USA |
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| Platform ID |
GPL16791 |
| Series (2) |
| GSE155022 |
dCas9 Fusion to Computer Designed PRC2 Inhibitor Reveals Functional TATA Box in Distal Promoter Region [bulk RNA-seq] |
| GSE195555 |
dCas9 Fusion to Computer Designed PRC2 Inhibitor Reveals Functional TATA Box in Distal Promoter Region |
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| Relations |
| BioSample |
SAMN15636392 |
| SRA |
SRX8816472 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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