|
| Status |
Public on Jun 06, 2021 |
| Title |
D5_CTCFbKO_DDSOX17+S_CXCR4+_Rep1 |
| Sample type |
SRA |
| |
|
| Source name |
Human induced pluripotent stem cell (hiPSC)
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: ZIP13K2
differentiation days: Day 5
perturbation: SOX17(∆5'CTCF) + transgenic rescue element(SOX17)
treatment: 1 uM Shield-1 treated
cxcr4 status: CXCR4+
|
| Treatment protocol |
PB-CAG-DD-3xFLAG-hSOX17-GS-TagBFP-BGHpA rescue construct was generated by Gibson Assembly® (NEB, E2621L) of BstBI /BamHI double-digested PB-CAG-BGHpA (Addgene Plasmid #92161) and EcoRI digested synthetically generated pUC19 DD-3xFLAG-SOX17-GS-TagBFP (Genewiz). Both, PB-CAG-DD-3xFLAG-hSOX17-GS-TagBFP-BGHpA and pNF21a-Piggy-BAC-Transposase vector were co-transfected into SOX17∆5’CTCF#8.2 mTeSR1 (Stemcell Technologies) maintained human induced pluripotent stem cells of the ZIP13K224 cell line harboring the SOX17 boundary 2 deletion. Transfection was conducted by the use of equimolar plasmid ratios in combination with Lipofectamine Stem Transfection Reagent (Thermo Fischer Scientific, STEM00003) according to the manufacturer’s instructions. Transfected or untransfected cells were treated with mTeSR1 (Stemcell Technologies) containing 250 µg/ml m Hygromycin B (Carl Roth, 1287.1) for 2 weeks. TagBFP negative surviving cells were FACS-sorted on the FACS Aria Fusion (Beckton Dickinson) and seeded in low density (5-10x105/10 cm) using mTeSR1 supplemented with 10 µM Y-27632 (Tocris, 1254) to derive a polygenic /polyclonal SOX17 rescue cell line. To stabilize ectopic SOX17-TagBFP protein, undifferentiated iPSC or day 2 dEN onwards differentiating cells were treated with 1 µM final Shield-1 (Takara, 632189) back to back with untreated controls before sample collection for downstream analysis.
|
| Growth protocol |
ZIP13K2 cultures were treated with Accutase (Sigma-Aldrich, A6964) supplemented by 10 µM Y-27632 (Tocris, 1254) to obtain single cells. To quench and wash the cells, equal volumes of mTeSR1 (Stemcell Technologies) were added and cells spun down for 5 min. at 300 x g, 21°C. After resuspension in mTeSR1 supplemented by 10 µM Y-27632, cells were counted and seeded according to the manufacturer’s instructions on Matrigel (Corning) pre-coated culture plates / dishes. Media change using the STEMdiff Trilineage Endoderm Differentiation media (Stemcell Technologies) was performed on a daily base after washing the cultures with equal volumes of DPBS (Thermo Fischer Scientific, 14190250) according to the manufacturer’s instructions. Triplicates of either undifferentiated or differentiated ZIP13K226 cultures were treated with Accutase® (Sigma-Aldrich, A6964) and differentiated cultures were further quenched with FACS-buffer containing 5 mM EDTA (ThermoFischer Scientific, 15575020) 10% Fetal bovine serum (FBS) (ThermoFischer Scientific, 26140079) in DPBS (Thermo Fischer Scientific, 14190250). In order to enrich for CXCR4- or CXCR4+ cell fractions of differentiated cultures, cells were stained for anti-Human CRCX4 (CD184) PE (as described under 21. FACS) and compared to Isotype and unstained control sorted for either CXCR4- or CXCR4+ on the Aria II (Beckton Dickinson).
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA was extracted using the RNeasy Mini Kit (Qiagen).
RNA isolation including on-column DNase digest of enriched cell populations was performed using the RNeasy Mini Kit (Qiagen, 74104) according to the manufacturer’s instructions.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Data processing |
Gene counts are quantified by using RSEM default parameters with REFSEQ annotation.
Genome_build: hg19
Supplementary_files_format_and_content: Gene quantification files (*.genes.results) are generated using RSEM
|
| |
|
| Submission date |
Jan 22, 2021 |
| Last update date |
Jun 07, 2021 |
| Contact name |
Hua-Jun Wu |
| E-mail(s) |
hjwu@pku.edu.cn
|
| Organization name |
Peking University Health Science Center
|
| Department |
School of Basic Medical Sciences
|
| Lab |
Center for Precision Medicine Multi-Omics Research
|
| Street address |
8 science park road, zhongguancun life science park
|
| City |
Beijing |
| State/province |
Beijing |
| ZIP/Postal code |
102206 |
| Country |
China |
| |
|
| Platform ID |
GPL20301 |
| Series (2) |
| GSE127196 |
Topological isolation as a mechanism for precise control of developmental regulators in mammalian genomes |
| GSE165339 |
Topological isolation as a mechanism for precise control of developmental regulators in mammalian genomes [RNA-seq II] |
|
| Relations |
| BioSample |
SAMN17496932 |
| SRA |
SRX9920647 |
