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| Status |
Public on Sep 21, 2017 |
| Title |
mESC_Serum_RB18 |
| Sample type |
SRA |
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| Source name |
Embryonic stem cells (ESCs)
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| Organism |
Mus musculus |
| Characteristics |
cell type: ESC
culture conditions: cultured in serum
passages: >10
strain: C57BL/6
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| Treatment protocol |
ESC lines were maintained at 37˚C in 5% CO2, cultivated on gelatinized tissue-culture plates and dishes (Sigma-Aldrich), and passaged every other day. Serum ESCs were cultured in Knockout™ DMEM (Invitrogen) supplemented with 15% ES-qualified fetal bovine serum (HyClone), 0.1 mM β-mercaptoethanol (Sigma-Aldrich), 0.1 mM non-essential amino acids (Invitrogen), 2 mM L-glutamine (Invitrogen), 100 U/ml penicillin, 100 μg/ml streptomycin (Invitrogen), and 1000 U/ml mouse LIF (mLIF, Royan Biotech). 2i and R2i cells were cultured in N2B27 media that consisted of Neurobasal® medium and DMEM/F-12 (both from Invitrogen) at a 1:1 ratio, 1% N2 supplement (Invitrogen), 1% B27 supplement (Invitrogen), 0.1 mM β-mercaptoethanol, 5 mg/ml BSA (Invitrogen), 2 mM L-glutamine, 0.1 mM non-essential amino acids, 100 U/ml penicillin, 100 μg/ml streptomycin, and 1000 U/ml mLIF. Small-molecule chemicals used in 2i and R2i cultures were added at the following concentrations: 1 μM PD0325901 (Stemgent), 10 μM SB431542 (Sigma-Aldrich), and 3 μM CHIR99021 (Stemgent).
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| Growth protocol |
ESC lines were initially derived from C57BL/6 mice using the R2i+LIF protocol (Hassani ei al., 2014). Isolated R2i cells were either maintained under 2i+LIF conditions or transferred to media containing serum+LIF. Cells were passaged at least 10 times to derive stable 2i and serum cell lines. Pluripotency of established cell lines was confirmed by chimera formation and germline contribution as previously reported (Hassani et al., 2014).
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from the mESC samples using miRVana miRNA Isolation Kit (Invitrogen)
Total RNA was extracted from the mESC samples and used to generate small RNA libraries from which cDNA libraries were prepared at LC Sciences. The purified cDNA libraries were then used for cluster generation on Illumina’s Cluster Station and then sequenced on Illumina GAIIx (Illumina HiSeq 1000/Illumina GAIIx) according to the vendor’s instruction for running the instrument. Raw sequencing reads (up to 45 nt) were obtained using Illumina’s Sequencing Control Studio software version 2.8 following real-time sequencing image analysis and base-calling by Illumina's Real-Time Analysis version 1.8.70 (LC Sciences).
Illumina Standard Small RNA sequencing library construction protocol
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| Library strategy |
miRNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Data processing |
Quality control with FastQC v0.11.5
Adapter removal with Cutadapt v1.3
Alignment with Bowtie v1.1.2
Abundance measurement with featureCounts v1.5
miARma processing pipeline v1.5
Genome_build: mm10
Supplementary_files_format_and_content: [FeatureCounts.txt] non-normalized abundance measurements
Supplementary_files_format_and_content: [NormalizedExpression.txt] Normalized expression levels (log2)
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| Submission date |
Sep 21, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Ali Sharifi-Zarchi |
| E-mail(s) |
asharifiz@gmail.com
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| Organization name |
Royan Institute for Stem Cell Biology and Technology
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| Street address |
Royan Institute, Bani Hashem Sq.
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| City |
Tehran |
| ZIP/Postal code |
P.O. Box: 16635-148 |
| Country |
Iran |
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| Platform ID |
GPL22473 |
| Series (1) |
| GSE87174 |
Genome-wide expression patterns of microRNAs in ground state embryonic stem cells |
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| Relations |
| SRA |
SRX2172367 |
| BioSample |
SAMN05783043 |
| 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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