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| Status |
Public on Dec 19, 2019 |
| Title |
Ecto Rep 3 |
| Sample type |
SRA |
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| Source name |
H9-derived hPSC differentiation culture
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| Organism |
Homo sapiens |
| Characteristics |
lineage day: 5
passage number: 4/44
experiment date: 5.30.17
rna extraction date: 5.30.17
lineage: Ecto
number of cells: Unknown
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| Treatment protocol |
Ectoderm Differentiation: 1.2 x 106 cells were seeded in each well of a 6 well plate in mTeSR1 media (Stemcell Technologies) with 10 μM ROCK inhibitor (Y-27632; Stemcell Technologies). Media was changed to differentiation media 24 hours after seeding. hPSCs were differentiated over a 5-day period by culturing in a 50% DMEM/F12 and 50% IMDM-based medium (Gibco) supplemented with 450 μM monothioglycerol (Millipore Sigma), 1 mg/ml BSA (Gibco), 0.11 μM 2-mercaptoethanol (Gibco), 1% Glutamax (Gibco), 1% N2 supplement (Thermofisher), 2% B27 supplement (Thermofisher), 10 μM SB43154 (Stemgent), and 0.2 μM Dorsomorphin (Stemgent). Mesoderm Differentiation: 1.8 x 106 cells were seeded in each well of a 6 well plate in mTeSR1 media with 10 μM ROCK inhibitor (Y-27632; Stemcell Technologies). hPSCs were differentiated over a 5-day period by culturing in a 50% DMEM/F12 and 50% IMDM-based medium (Gibco) supplemented with 450 μM monothioglycerol (Millipore Sigma), 1 mg/ml BSA (Gibco), 0.11 μM 2-mercaptoethanol (Gibco), 1% Glutamax (Gibco), 0.7 µg/mL insulin (Millipore Sigma), 15 µg/mL transferrin (Millipore Sigma), 1 mL/100mL chemically-defined lipid concentrate (Gibco), 100 ng/mL VEGF-165 (Stemcell Technologies), 100 ng/mL BMP4 (Peprotech), and 20 ng/mL FGF2 (Peprotech). Medium for the first 24 hours of differentiation was supplemented with 100 ng/mL Activin A (Stemcell Technologies). Endoderm Differentiation: 1.8 x 106 cells were seeded in each well of a 6 well plate in mTeSR1 media with 10 μM ROCK inhibitor (Y-27632; Stemcell Technologies). hPSCs were differentiated over a 3-day period by culturing in a 50% DMEM/F12 and 50% IMDM-based medium (Gibco) supplemented with 450 μM monothioglycerol (Millipore Sigma), 1 mg/ml BSA (Gibco), 0.11 µM 2-mercaptoethanol (Gibco), and 1% Glutamax (Gibco). Medium for the first 24 hours was supplemented with 100 ng/mL Activin A (Stemcell Technologies), 2 µM CHIR99021 (Cayman Chemicals), and 50 nM PI-103 (Fisher Scientific). Medium after the first 24 hours was supplemented with 100 ng/mL Activin A (Stemcell Technologies), and 250 nM LDN 1931189 (Stemgent). Embryoid Body Differentiation : 3.5 x 106 cells per well were seeded in AggreWell EB Formation Media (Stemcell Technologies) with 10 μM ROCK inhibitor (Y-27632; Stemcell Technologies) into 6-well AggreWell 400 plates (Stemcell Technologies) for consistent size and shape. Within 24 hours, embryoid bodies were harvested through a cell strainer and transferred to 6-well Ultra-Low Attachment plates (Corning Costar) and grown in DMEM/F12 (Gibco) supplemented with 20% KnockOut Serum Replacement (Gibco), 1% Glutamax (Gibco), 1% Penicillin/Streptomycin (Corning), 1% Non-Essential Amino Acids (Corning), and 0.1 mM 2-mercaptoethanol (Gibco). Media was changed every day throughout 21 days of differentiation. For DCA treated condition, media was supplemented with 1mM dichloroacetate (DCA; Millipore Sigma). For glutamine deprivation condition, DMEM/F12 without glutamine (Gibco) was used and Glutamax (Gibco) was excluded from media preparation.
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| Growth protocol |
All work with human embryonic stem cells (hESCs) has been approved under the UCLA Institutional Biosafety Committee (IBC) and UCLA Embryonic Stem Cell Research Oversight (ESCRO) Committee under Protocol # 2007-003-12. H9 (WA09 – Female; RRID:CVCL_9773), H1 (WA01 – Male; RRD:CVCL_9771), HSF-1 (Male; RRID:CVCL-D003), hiPS2 (Male; RRID: CVCL_B508) and UCLA-1 (Female; RRID:CVCL_9951) primed human pluripotent stem cells (hPSCs) were provided low-passage and contamination tested through the UCLA BSCRC hESC Core Bank (Jerome Zack, UCLA). Following receipt, hPSCs were shifted to feeder-free Matrigel (Corning) in mTeSR1 medium (Stemcell Technologies) and passaged using Gentle Cell Dissociation Reagent (Stemcell Technologies). 6-wells plates were coated with 1:10 Matrigel (Corning) and incubated for 30 minutes prior to seeding. 80-90% confluent hPSCs were incubated in Gentle Cell Dissociation Reagent (Stemcell Technologies) for 8 minutes at 37C, dissociated to single cells using a 40μm cell strainer, and harvested in plain media (DMEM/F12; Gibco) for counting. Cells were centrifuged at 450xg for 5 minutes.
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| Extracted molecule |
total RNA |
| Extraction protocol |
All cells were grown to 70-80% confluence and purified using the RNeasy Mini Kit (Qiagen) and RNase-free DNase (Qiagen) following the manufacturer’s protocols. All samples showed a A260/280 ratio > 1.99 (Nanodrop; Thermo Scientific). For RNA-Sequencing, prior to library preparation, quality control of the RNA was performed using the Advanced Analytical Technologies Fragment Analyzer (Advanced Analytical, Inc.) and analyzed using PROSize 2.0.0.51 software. RNA Quality Numbers (RQNs) were computed per sample between 8.1 and 10, indicating intact total RNA per sample prior to library preparation.
Strand-specific ribosomal RNA (rRNA) depleted RNA-Seq libraries were prepared from 1 µg of total RNA using the KAPA Stranded RNA-Seq Kit with Ribo-Erase (Kapa Biosystems, Roche). Briefly, rRNA was depleted from total RNA samples, the remaining RNA was heat fragmented, and strand-specific cDNA was synthesized using a first strand random priming and second strand dUTP incorporation approach. Fragments were then A-tailed, adapters were ligated, and libraries were amplified using high-fidelity PCR. All libraries were prepared in technical duplicates per sample (n = 30 samples, 60 libraries total), and resulting raw sequencing reads merged for downstream alignment and analysis. Libraries were paired-end sequenced at 2x150 bp on an Illumina NovaSeq 6000.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
RS11
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| Data processing |
Fibroblasts, iPSCs, and MSCs were each sequenced in biological triplicates and technical duplicates (n = 60 total samples) to account for variation in extraction and culturing. Raw sequencing reads were converted into fastq files and filtered for low quality reads and Illumina sequencing adapter contamination using bcl2fastq (Illumina). Reads were then quasi-mapped and quantified to the Homo sapiens GENCODE 28 (GRCh38.p12, Ensembl 92, April 2018) transcriptome using the alignment-free transcript level quantifier Salmon v0.9.1 8-10. A quasi-mapping index was prepared using parameters “salmon index -k 31 –type quasi”, and comprehensive transcript level estimates were calculated using parameters “salmon quant -l A –seqBias –gcBias --discardOrphansQuasi”. Transcript level counts were collapsed to gene level (HGNC) counts, transcripts per million abundances (TPM) and estimated lengths using R Bioconductor package tximport v1.6.0 11.
The resulting sample gene count matrix was size factor normalized and analyzed for pairwise differential gene expression using R Bioconductor package DESeq2 v1.18.1. Expression changes were estimated using an empirical Bayes procedure to generate moderated fold change values with design “~ Batch + Sample”, modeling batch effect variation due to day of RNA extraction 12,13. Significance testing was performed using the Wald test, and resulting P values were adjusted for multiple testing using the Benjamini-Hochberg procedure 14. DEGs were filtered using an adjusted false discovery rate (FDR) q value < 0.05 and an absolute log2 transformed fold-change > 1.
Variance stabilized transform (VST) values in the gene count matrix were calculated and plotted for PCA using R Bioconductor packages DESeq2, FactoMineR, and factoextra 12,13. Whole transcriptome PCA was performed with comparison to existing RNA-sequencing data of nutrient balanced trilineage differentiations previously provided in GEO: GSE101655 1. RNA-sequencing samples were batch effect normalized across library preparation differences between this study and previously published reports 1 using limma v3.34.9 15. PCA of nucleus-encoded mitochondrial protein and mtDNA transcripts were extracted using localization evidence derived from MitoMiner v4.0, subsetting VST matrices using genes listed in MitoCarta 2.0 16,17. Scatterplots and MA plots of gene expression fold-changes between fibroblasts, iPSCs, and MSCs were performed, and Pearson/Spearman correlation coefficients calculated, using R package ggpubr v0.1.6 (https://cran.r-project.org/web/packages/ggpubr/index.html). Genes of interest were extracted and averaged clonal heatmaps were prepared using R Bioconductor packages pheatmap v1.0.8 and gplots v3.0.1 18,19. Venn diagram intersections of DEG lists were generated using Venny 2.1.0 (http://bioinfogp.cnb.csic.es/tools/venny/index.html)
Genome_build: Homo sapiens GENCODE 28 (GRCh38.p12, Ensembl 92, April 2018)
Supplementary_files_format_and_content: Salmon v0.9.1 quantification output files for each respective sample after quasi-mapping to the GENCODE 28 reference transcriptome. Columns are (in order): (1) Name of transcript from GENCODE 28 reference, (2) Length of the transcript (nt), (3) Effective length of the transcript, correcting for sequencing and GC fragment bias, (4) transcripts per million of the transcript (TPM), and (5) number of estimated reads mapping to each quantified transcript.
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| Submission date |
Feb 27, 2019 |
| Last update date |
Dec 19, 2019 |
| Contact name |
Fasih Mubtasim Ahsan |
| Organization name |
Massachusetts General Hospital
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| Department |
Center for Genomic Medicine
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| Lab |
Soukas Lab
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| Street address |
185 Cambridge Street, CPZN 6500
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02114 |
| Country |
USA |
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| Platform ID |
GPL24676 |
| Series (1) |
| GSE127270 |
Transcriptional profiling of nutrient-balanced early trilineage differentiations derived from H9 hPSCs. |
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| Relations |
| BioSample |
SAMN11029444 |
| SRA |
SRX5442055 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3633420_RS11_salmon.quant.sf.txt.gz |
3.3 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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