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| Status |
Public on May 31, 2018 |
| Title |
human_ESC_w3_fj105 |
| Sample type |
SRA |
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| Source name |
embryonic stem cells
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| Organism |
Homo sapiens |
| Characteristics |
cell line: H9
time point: week 3
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| Growth protocol |
The Eiraku et al., 2008 protocol was optimized for use with human ESCs, rhesus ESCs, chimpanzee iPSCs, and orangutan iPSCs. Human H9 and rhesus LyonESC1 embryonic stem cells were cultured on mouse embryonic fibroblasts with KSR-8 media (KO DMEM/F-12 + 20% KOSR, 1% NEAA, 1% GlutaMAX, 1% Pen-strep, and 0.1mM 2-mercaptoethanol supplemented with 8ng/mL bFGF). Embryonic stem cells were manually lifted from MEF feeders and allowed to self-form into embryoid bodies on low attachment plates (Corning) in KSR media. Chimpanzee and orangutan induced pluripotent stem cells were grown in feeder-free conditions on matrigel (Corning) with mTeSR-1 (Stem Cell Tech) or vitronectin (ThermoFisher) with Essential-8 Flex media (ThermoFisher), respectively, and 10,000 cells per EB were aggregated using AggreWell-800 plates (Stem Cell Technologies) in Aggrewell media (Stem Cell Technologies) supplemented with 10 uM Y-27632 rock inhibitor (ATCC) and transferred to low attachment plates (Corning) on day 2. Both methods supplemented the respective media with 500ng/mL DKK1 (Peprotech), 500 ng/mL NOGGIN (R & D Systemes), 10 uM SB431542 (Sigma), and 1 uM Cyclopamine V. californicum (VWR) for the first 18 days of differentiation. The media was changed to Neuralbasal (Invitrogen) supplemented with N2 (Gibco) and 1 uM Cyclopamine on day 18. At this time, chimpanzee and orangutan neurospheres were also supplemented with 10ng/mL bFGF and 10ng/mL EGF to improve survivability in Neuralbasal media. After day 26, all cultures were grown in Neuralbasal/N2 media without any added factors. Total RNA was extracted at weekly time points for each species. This timeline was adjusted accordingly in rhesus, harvesting on days 6, 11, 17, 22, and 28, to account for differences in gestational timing.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol + RNeasy or Direct-zol cleanup, followed by ribozero rRNA depletion.
Strand-specific dUTP followed by TruSeq DNA library prep kit.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
fj105
Processed data file: hg19.RSEM.gene_expression.txt
Processed data file: deseq_baseMean_allF2.txt
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| Data processing |
Paired-end Illumina reads were trimmed from the 3’ end of read1 and read2 to 100x100bp for human and rhesus libraries and 80x80bp for chimpanzee and orangutan based on sequence quality. Bowtie2 v2.2.1 was used with the “--very-sensitive” parameter to filter reads against the repeatMasker library for each respective species which were removed from further analysis.
STAR v2.5.1b was used to map RNA-seq reads to hg19 (human, Genome Reference Consortium GRCh37, 2009), panTro4 (chimpanzee, CGSC Build 2.1.4, 2011), ponAbe2 (orangutan, WUSTL Pongo_albelii-2.0.2, 2007), and rheMac8 (rhesus macaque, Baylor College of Medicine HGSC Mmul_8.0.1, 2015) respective to the origin species. STAR was run with the default parameters with the following exceptions: --outFilterMismatchNmax 999, --outFilterMismatchNoverLmax 0.04, --alignIntronMin 20, --alignIntronMax 1000000, and --alignMatesGapMax 1000000. STAR alignments were converted to genomic position coverage with the bedtools command genomeCoverageBed -split.
DESeq2 v1.14.1 was used to provide basemean expression values and differential expression analysis across the time course in each species using FANTOM5 lv3 (Hon et al., 2017) transcript annotations (lifted to other species by transMap). Total gene coverage for a gene was converted to read counts by dividing the coverage by N+N (100+100 for human and rhesus and 80+80 for chimpanzee and orangutan) since each paired-end NxN mapped read induces a total coverage of N+N across its genomic positions.
Cufflinks v2.0.2 suite was used to assemble transcript predictions of potentially unannotated lncRNAs in each species and the Cuffmerge tool was used to combine these annotations with FANTOM5 transcripts.
Cufflinks-assembled and merged transcript sets were then projected through the cactus alignment to each of the other three genomes (hg19, panTro4, ponAbe2, and rheMac8, respectively).
RSEM v1.3.0 was used to provide TPM expression values for Cufflinks generated transcripts.
Genome_build: hg19, panTro4, ponAbe2, and rheMac8
Supplementary_files_format_and_content: *tsv, *txt: Tab-delimited text files include either basemean values (DESeq) or TPM values (RSEM) for each time point in each of the respective species.
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| Submission date |
Oct 26, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Andrew Ryan Field |
| E-mail(s) |
andrew_field@hms.harvard.edu
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| Organization name |
Harvard Medical School
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| Department |
BCMP
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| Lab |
Adelman
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| Street address |
45 Shattuck St
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
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| Platform ID |
GPL11154 |
| Series (1) |
| GSE106245 |
Human embryonic stem cell, chimpanzee induced pluripotent stem cell, orangutan induced pluripotent stem cell, rhesus embryonic stem cell, and their derived cortical organoid RNA-seq |
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| Relations |
| BioSample |
SAMN07839661 |
| SRA |
SRX3333998 |
| 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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