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| Status |
Public on Nov 01, 2018 |
| Title |
BOFS p2hNCC |
| Sample type |
SRA |
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| Source name |
BOFS p2hNCC
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| Organism |
Homo sapiens |
| Characteristics |
cell line: BOFS hiPSC
genotype: BOFS 88Mb inversion
treatment: hiPSC differentiation into hNCC (passage 2)
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| Treatment protocol |
BOFS hiPSC were derived from fibroblasts isolated from a BOFS patient carrying a 88Mb de novo heterozygousinversion in chromosome 6.
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| Growth protocol |
WT hiPSC and BOFS hiPSC lines were grown in tissue culture plates coated with GelTrex (ThermoFisher Scientific, #1413301) using either mTeSRTM1 (STEMCELL Technologies, #05870) or StemMACS iPS-Brew XF medium (Miltenyl Biotec, #130-104-368). The medium of the cells was changed daily. hiPSC were differentiated into hNCC experiments as previously reported (Bajpai et al;2010). Briefly, hiPSCs were washed twice with PBS and treated with 2mg/mL Collagenase solution (Life Technologies, #17104-019) in DMEM KO Medium to detach hiPSC colonies from the plate surface. 2mL of Collagenase solution was used for one well of a 6-well plate, followed with incubation period at 37C. After about one hour, collagenase solution was carefully aspirated and PBS was added to collect all the colonies into a 15mL falcon tube. Following another round of PBS washing, fresh pre-warmed hNCC Differentiation Medium was added and resuspended to break big colonies into relatively smaller cell clumps. hiPSC clumps were then plated in petri dishes and kept at 37⁰C, which after 2-3 days form floating Embryoid Bodies (EB) formation . Between day 6 and 9, EBs attached and gave rise to hNCC outgrowths. hNCC were collected at day 11 (d11hNCC) and then expanded for two passages (p2hNCC) according to a recent protocol (Prescott et al; 2015). The hNCC differentiation medium was changed every 2-3 days.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Cells were dissociated to generate a suspension of viable single cells. The suspension was centrifuged at 300 rcf for 5 min and the cell pellet was washed in 1 ml of 1xPBS 0.04% BSA. Cells were centrifuged again and resuspended in 1xPBS 0.04% BSA to the desired cell concentration. Cells were passed throught strainer and cell concentration was determined again. cDNA synthesis and library preparation was performed following the 10x Single Cell 3’ Reagent Kit protocol on a 10xGenomics Chromium instrument. Briefly, cells were partitioned in nanoliter-scale Gel Bead-In-Emulsions (GEMs) containing, in addition to a single cell, a master mix and primers that consist of (i) an Ilumina R1 sequence, (ii) a 16bp 10x barcode unique for each cell, (iii) a 10 bp Unique Molecular Identifier (UMI) and (iv) a poly-dT sequence. Full length barcoded cDNA were generated from poly-adenylated mRNAs. GEMs were broken and the pooled fractions were recovered.
The cDNAs were exposed to enzymatic fragmentation and size selection prior to library construction and P5, P7, a sample index and an Ilumina R2 primer were added during library construction. The final libraries containing the P5 and P7 primers were used for Ilumina bridge amplification. Finally, R1 and R2 primers were used for paired-end Ilumina sequencing.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
UMIs were counted using cellranger-2.1.0 with default parameters on hg19.
Initial dimensionality reduction, clustering and visualization was generated with cellranger-2.1.0 and cellrangerRkit_2.0.0 within R-3.4.0.
Counts were aggregated into a single matrix with default normalization (“--normalize=mapped”) by cellranger-2.1.0.
Matrix was further processed with monocle_2.6.4 within R-3.4.0.
Dropouts were corrected with Rmagic_1.3.0 (MAGIC) within R-3.4.0 for correlation analysis.
Genome_build: hg19
Supplementary_files_format_and_content: File to visualize gene expression in all the sequenced single cells using the Luope Cell Browser software (10xGenomics)
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| Submission date |
Oct 17, 2018 |
| Last update date |
Nov 01, 2018 |
| Contact name |
Milos Nikolic |
| E-mail(s) |
milosn@gmail.com
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| Organization name |
Center for Molecular Medicine Cologne
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| Street address |
Robert Koch Str. 21
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| City |
Koeln |
| State/province |
Nordrhein-Westfalen |
| ZIP/Postal code |
50674 |
| Country |
Germany |
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| Platform ID |
GPL16791 |
| Series (2) |
| GSE108522 |
Disruption of the TFAP2A regulatory domain causes Branchio-Oculo-Facial Syndrome (BOFS) and illuminates pathomechanisms for other human neurocristopathies |
| GSE121429 |
Disruption of the TFAP2A regulatory domain causes Branchio-Oculo-Facial Syndrome (BOFS) and illuminates pathomechanisms for other human neurocristopathies [scRNA-seq] |
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| Relations |
| BioSample |
SAMN10254704 |
| SRA |
SRX4901062 |
| 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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