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Sample GSM3436240 Query DataSets for GSM3436240
Status Public on Nov 01, 2018
Title BOFS p2hNCC
Sample type SRA
Source name BOFS p2hNCC
Organism Homo sapiens
Characteristics cell line: BOFS hiPSC
genotype: BOFS 88Mb inversion
treatment: hiPSC differentiation into hNCC (passage 2)
Treatment protocol BOFS hiPSC were derived from fibroblasts isolated from a BOFS patient carrying a 88Mb de novo heterozygousinversion in chromosome 6.
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.
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.
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2500
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)
Submission date Oct 17, 2018
Last update date Nov 01, 2018
Contact name Milos Nikolic
Organization name Center for Molecular Medicine Cologne
Street address Robert Koch Str. 21
City Koeln
State/province Nordrhein-Westfalen
ZIP/Postal code 50674
Country Germany
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]
BioSample SAMN10254704
SRA SRX4901062

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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