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| Status |
Public on Jul 03, 2014 |
| Title |
miRNA-seq_R-NSCP6 |
| Sample type |
SRA |
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| Source name |
R-NSCs at late passages
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| Organism |
Macaca mulatta |
| Characteristics |
developmental stage: The sixth passage of rosettes culture
genotype: wild type
cell line: IVF3.2
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| Growth protocol |
Rhesus monkey ESCs (line IVF3.2) were cultured on feeder cells of mitotically inactivated mouse embryonic fibroblasts (MEFs) as described previously. Embryoid bodies (EBs) differentiation was employed to induce neural differentiation. Briefly, rESC colonies were transferred onto tissue culture dishes coated with 1% agar and differentiated in ESC culture medium depleted of bFGF to induce EB differentiation. EBs were then transferred onto ECM-coated (10 μg/cm2, Sigma-Aldrich) tissue culture dishes to spread and propagate in DMEM/F12 medium (Gibco) supplied with ITS (Sigma-Aldrich), 2.5 ug/mL fibronectin (Millipore), 60 µM putrescine (Sigma-Aldrich), and 20 ng/ml bFGF. Neural tube-like structures could be observed in EB outgrowth and rosette-like clusters were then manually separated and expanded according to the protocol described by Elkabetz et al. with minor modifications. In brief, rosette clusters were replaced on culture dishes pre-coated with 15 µg/mL polyornithine plus 1 µg/mL laminin (Po/Lam) (Sigma-Aldrich) in N2 medium supplemented with SHH (200 ng/mL; R&D), FGF8 (100 ng/mL; Sigma-Aldrich), ascorbic acid (0.2 mM; Sigma-Aldrich), and brain-derived neurotrophic factor (BDNF, 20 ng/mL; R&D). The first passage of rosettes culture was defined as R-NSCP1. Cells were passed at density of 100–400 × 103 cells/cm2 when the growth reached 80% confluency. R-NSCs were propagated in Po/Lam culture dishes in N2 medium with 0.2 mM ascorbic acid, 20 ng/mL BDNF, 500 ng/mL SHH, 500 ng/mL Dll4 (R&D) and 500 ng/mL Jagged-1 (R&D). Under this culture condition, rosette structure can be maintained for up to five generations. At the 6th generation, the rosette structure was no longer visible and the cells were defined as R-NSCP6. Neural progenitor cells (NPCs) were derived from R-NSCs with extended culture on Po/Lam culture dishes in N2 medium plus 0.2 mM ascorbic acid, 20 ng/mL BDNF, 20 ng/mL FGF2, and 20 ng/mL EGF for more than 100 days.
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| Extracted molecule |
total RNA |
| Extraction protocol |
According to the manufacturer’s instructions (illumina), mRNA was purified using poly-T oligo-attached magnetic beads. Following purification, the mRNA was fragmented into small pieces. The cleaved RNA fragments were converted to the first strand cDNA followed by generation of double strand cDNAs. These cDNA fragments went through end repair course by adding a single ‘A’ base as well as the adapter ligation. For miRNA sequencing, total RNA was ligated to a pair of adaptors at the 5’ and 3’ends according to the manufacturer’s instructions (Illumina). RNA molecules were converted to cDNA and amplified by RT-PCR using adaptor primers.
The libraries prepared from samples of ESCs, R-NSCP1, R-NSCP6, and NPCs were used for paired-end mRNA sequencing by Illumina HiSeq 2000 and single-end miRNA sequencing by Illumina HiSeq 2000.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
Supplementary S2.xls
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| Data processing |
RNA-seq reads were aligned to the rheMac3 genome assembly using Bowtie 0.12.8.0
Tophat 2.0.5 was used to analyze the mapping results to identify splice junctions
Cufflinks 2.0.2 were used to assemble transcripts, estimate expression abundances, and test for differentially expressed genes between stages
miRNA-seq reads were aligned to rhesus rheMac3 and miRBase (version 19) using miRDeep2
Alternative Splicing analyses was performed using MATS software (version 3.0.7.beta)
Genome_build: rheMac3
Supplementary_files_format_and_content: Microsoft Excel files, with rows representing genes and columns representing stages. The values are FPKM values in mRNA profile (Supplementary S1.xls) and the values are read counts in miRNA-seq (Supplementary S2.xls).
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| Submission date |
Dec 12, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Yuqi Zhao |
| E-mail(s) |
zhaoyuqi616@ucla.edu
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| Organization name |
University of California, Los Angeles
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| Department |
Department of Integrative Biology and Physiology
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| Street address |
2000 Terasaki Life Sciences Bldg 610 Charles E. Young Drive East
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| City |
Los Angeles |
| State/province |
CA |
| ZIP/Postal code |
90095 |
| Country |
USA |
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| Platform ID |
GPL14954 |
| Series (1) |
| GSE53260 |
mRNA-Seq and microRNA-Seq whole-transcriptome analysis of rhesus monkey ESC neural differentiation revealed the potential rosette neural stem cell regulators |
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| Relations |
| BioSample |
SAMN02442678 |
| SRA |
SRX390824 |
| Supplementary data files not provided |
SRA Run Selector |
| Processed data are available on Series record |
| Raw data are available in SRA |
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