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| Status |
Public on Jan 11, 2023 |
| Title |
Day0_undifferentiated_iPSCs_bulk_RNAseq_rep1 |
| Sample type |
SRA |
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| Source name |
Induced pluripotent stem cells
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| Organism |
Homo sapiens |
| Characteristics |
tissue: Induced pluripotent stem cell
cell line: WTC-11 (GM25256)
age group: day0
treatment: No treatment
experiment type: bulk RNA-seq
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| Biomaterial provider |
Coriell Cell Repositories http://ccr.coriell.org/Sections/Search/Search.aspx?PgId=165&q=GM25256
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| Treatment protocol |
Briefly, hiPSCs (WTC-11 human induced pluripotent stem cells) (Coriell, #GM25256) were seeded on 12-well plates coated with growth factor-reduced Matrigel (Corning) and cultured in mTeSR1 stem cell medium (stemcell, #85850) until cells reach confluency with medium changes daily. On the first day of differentiation (deemed Day 0), stem cell media is replaced with a base media consisted of RPMI 1640 Medium (Thermo, #11875093) mixed with EpiLife (Thermo, #MEPI500CA) at 1:1 ratio, supplemented with 0.1x supplement S7 (Thermo, #S0175), 0.1uM β-mercaptoethanol (BME) (Sigma, #M7522) and 400um smoothened agonist (SAG) (Selleckchem, # S7779). At day 3 of differentiation 150pM of bone morphogenic protein-4 (BMP4) (rndsystems, #314-BP-010) is continuously added daily till day 7. At day 8, the base media is supplemented with 1uM of BMP-I inhibitor (LDN-193189)(Tocris, # 6053), 5uM of GSK3-Inhibitor (CHIR99021) (Selleckchem, # 4423), 500pM epidermal growth factor (EGF) (rndsystems, #236-EG) and 3.5μM of Neurotrophin-4 (NT4) (rndsystems, #268-N4). The cultures were then harvested at day 10 at an oral epithelium stage, or extended to day 16 by adding 300pM BMP4, and 800nM transforming growth factor beta 1(TGF-β1) (rndsystems, #7754-BH) for the pre-ameloblast stage at day16.
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| Growth protocol |
cells were grown in mTeSR1 stem cell media
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| Extracted molecule |
total RNA |
| Extraction protocol |
Frozen tissues were carefully transferred to a stack of chilled aluminum foil kept on dry ice to prevent thawing. The folded foil encapsulating the tissues were placed on a block of dry ice and the foil was pounded with a pestle to pulverize the tissues into powder. 1mL of lysis buffer that contains nuclei buffer (10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2, pH 7.4), 0.1% IGEPAL CA-630, 1% SUPERase In RNase inhibitor (20 U/μL, Thermo), and 1% BSA (20 mg/mL, NEB) were added onto the tissue powder and transferred to a 1.5mL tubes. Samples were incubated in the lysis buffer for 1 hour on ice. The samples were pipetted up and down with pre-cut 1000uL pipette tip to further disassociate the tissue. The dissociated tissues were passed through 70 um cell strainers (Corning) into a 50mL conical tube. The strainers were rinsed with lysis buffer to minimize nuclei loss. The samples were centrifuged to pellet the nuclei at 500g for 5 minutes at 4°C and the supernatant was discarded. The samples resuspended again in 1ml lysis buffer, transferred into new 15mL tubes, pelleted again and supernatant was discarded. The pellets were resuspended in 50ul of nuclei buffer, and 5 mL of 4% Paraformaldehyde (PFA) (EMS) diluted in RNase free PBS, was added to fix the nuclei for 15 minutes on ice. The tubes were flicked gently every 5 minutes to reduce clumping of nuclei. The fixed nuclei were pelleted at 500g for 3 minutes at 4°C and the PFA waste was discarded. The pelleted nuclei washed in nuclei wash buffer (cell lysis buffer without IGEPAL) and then centrifuged again at 500g for 5 minutes 4°C, and the supernatant was discarded. Finally, the pellets were resuspended again in nuclei wash buffer and then flash-frozen in liquid nitrogen before storing in –80°C.
For nuclei extraction from the differentiation culture, the cells were treated with StemPro Accutase (Theromo, #A1110501) for 7min to detach the cells and transfer them into 15mL tube, then incubated in trypsin (Theromo, #25300054) for another 7min to prevent re-clumping. The cells were span down to remove trypsin after inactivation with more media. The pellet was treated with nuclei lysis buffer and the same steps for nuclei extraction protocol were followed.
Single-cell combinatorial-indexing RNA-sequencing (sci-RNA-seq) protocol is described previously and more details can be found in this link (https://github.com/bbi-lab/). sci-RNA-seq relies on the following steps, (i) thawed nuclei were permeabilized with 0.2% TritonX-100 (Sigma, #T9284) (in nuclei wash buffer) for 3 min on ice, and briefly sonicated to reduce nuclei clumping; (ii) nuclei distributed across 96-well plates; (iii) A first molecular index is introduced to the mRNA of cells within each well, with in situ reverse transcription (RT) incorporating the unique molecular identifiers (UMIs); (iv) All cells were pooled and redistributed to multiple 96-well plates in limiting numbers (e.g., 10 to 100 per well) and a second molecular index is introduced by hairpin ligation;(v) Second strand synthesis, tagmentation, purification and indexed PCR; (vi) Library purification and sequencing is performed.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 2000 |
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| Data processing |
All libraries were sequenced on one NovaSeq platform (Illumina). The recipe used was: read 1: 34 cycles, read 2: 100 cycles, index 1: 10 cycles, index 2: 10 cycles. Base calls, downstream sequence processing and single-cell digital-expression matrix generation steps were similar to what was described in sci-RNA-seq3 paper17. STAR v.2.5.2b54 aligner used with default settings and gene annotations (GRCh38-primary-assembly, gencode.v27). Uniquely mapping reads were extracted, and duplicates were removed using the UMI sequence, reverse transcription index, hairpin ligation adaptor index and read 2 end-coordinate (that is, reads with identical UMI, reverse transcription index, ligation adaptor index and tagmentation site were considered duplicates).
All low-quality reads were removed from the data (including jaws, tooth germs and salivary glands samples from all time points) by setting UMI cutoff to 100 and removing all mitochondrial reads. Following Monocle3 workflow, data underwent normalization by size factor, preprocessing, dimension reduction (UMAP algorithm), and unsupervised graph-based clustering analysis (Leiden Algorithm). Certain clusters from the initial analysis were selected for further sub-clustering, and the previous analysis repeated. Pseudotime analysis also was done with Monocle3 following the default workflow, which include learning the graph, ordering the cells, and plotting the trajectory over UMAP. Mutual nearest neighbors (MNNs) algorithm was used for patch effect correction only between day10 differentiation sample and day16 sample. PanglaoDB, a curated single cell gene expression database was utilized to explore the consensus of cell type markers used across publicly available single cell datasets.
Genome_build: GRCh38-primary-assembly, gencode.v27
Supplementary_files_format_and_content: processed files contain cluster annotations, gene annotations in csv fromat. And the count matrix is in MatrixMarket format.
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| Submission date |
Sep 24, 2021 |
| Last update date |
Jan 11, 2023 |
| Contact name |
Hannele Ruohola-Baker |
| E-mail(s) |
hannele@uw.edu
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| Phone |
(206) 543-8468
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| Organization name |
University of Washington
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| Department |
Department of Biochemistry
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| Lab |
Ruohola-Baker
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| Street address |
UW Medicine at South Lake Union, 850 Republican Street
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| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98109 |
| Country |
USA |
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| Platform ID |
GPL30173 |
| Series (1) |
| GSE184749 |
Human iPSC Derived Enamel Organoid Guided by Single Cell Atlas of Human Tooth Development |
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| Relations |
| BioSample |
SAMN21611945 |
| SRA |
SRX12340621 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5596833_Day0_undifferentiated_bulk_RNAseq_rep1_count.txt.gz |
4.9 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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