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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jun 30, 2015 |
| Title |
ESC_shHEB_rep2 |
| Sample type |
SRA |
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| Source name |
Embryonic stem cells
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| Organism |
Mus musculus |
| Characteristics |
strain: CGR8
passages: 18-25
activin treatment: no
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| Treatment protocol |
At day 2, human Activin A (100 ng/ml; R&D systems) was added to induce endoderm differentiation without dissociation/reaggregation.
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| Growth protocol |
Mouse embryonic stem cells (ESCs) were cultured on gelatin coated plates in the absence of feeder cells. ESC medium was prepared by supplementing knockout DMEM (Invitrogen) with 15% FBS, 1 mM glutamax, 0.1 mM nonessential amino acids, 0.1 mM 2-mercaptoethanol and 1000 units of LIF (Millipore). To inhibit TGF-beta signaling pathway, ESCs were treated with 10 µM SB431542 (Tocris Bioscience) for 24 hours. For defined embryoid body (EB) differentiation, ESCs were dissociated by TrypLE Express and cultured in serum-free defined differentiation media consisting of 75% Iscove’s modified Dulbecco’s medium and 25% Ham’s F12 media supplemented with 0.5x of both N2 and B27 (without retinoic acid), 0.05% BSA and 50 µg/ml ascorbic acid (Sigma).
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA was isolated using the RNeasy Plus Mini Kit (Qiagen), and messenger RNA (mRNA) was purified from the total RNA using Dynabeads Oligo(dT)25 (Invitrogen) according to the manufacturer's instructions.
RNA sequencing libraries were built using an approach which targets the 3′ end (3SEQ) as described previously (Beck et al., 2010). Heat sheared mRNA was reverse transcribed with the P7_oligo-dT Index primers. The P5 linker was then ligated to the free end, and the linker-ligated cDNA was size-selected for 200-350 bp fragments by 3% Nusieve agarose gels (Lonza). The selected DNA was amplified using primers P5 and P7_Index for 15 cycles, and the amplified product was second size-selected for 200-350 fragments. The purified library quality was assessed using Bioanalyzer and Qubit, and sequenced on the Illumina HiSeq 2000 with multiplexing.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Basecalls performed using CASAVA version 1.8.2
Read sequences were trimmed to remove the 3SEQ adapters and leading or trailing low-quality bases using Trimmomatic 0.32 with parameters ILLUMINACLIP 2:30:5, LEADING:3, TRAILING:3
Trimmed reads were aligned by STAR 2.3.0e to the mm9 reference genome and a reference transcriptome prepared by combining Ensembl gene annotations with ENCODE RNA-seq data from E14 mouse ESCs using Cufflinks 2.2.1; candidate splice junctions that were both non-canonical and unannotated were discarded
Reads were counted in the last 1 kb of each transcript by the featureCounts tool in Subread 1.4.5,
Transcript read counts were tested for significant differences between HEB and control shRNA by DESeq2 1.2.8, analyzing each cell/treatment group indepedently; transcripts with no reads from any library were ignored
Genome_build: mm9
Supplementary_files_format_and_content: transcripts.gtf: transcript annotations from Ensembl + ENCODE RNA-seq, used for read alignment
Supplementary_files_format_and_content: transcripts_last1000.gtf: last 1 kb of each transcript annotation, used for read counting
Supplementary_files_format_and_content: transcript_counts.tsv: read counts from each library in each transcript's last 1 kb
Supplementary_files_format_and_content: DESeq2_analysis_*.tsv: results of DESeq2 analysis, including significance test and regularized log transformation of read counts
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| Submission date |
Aug 11, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Se-Jin Yoon |
| E-mail(s) |
sjyoon@stanford.edu
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| Phone |
650-736-0278
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| Organization name |
Stanford University
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| Department |
Genetics
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| Street address |
300 Pasteur Drive, Alway M311
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| City |
Stanford |
| State/province |
CA |
| ZIP/Postal code |
94305 |
| Country |
USA |
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| Platform ID |
GPL13112 |
| Series (2) |
| GSE60285 |
HEB associates with PRC2 and SMAD2/3 to regulate developmental fates [RNA-Seq] |
| GSE60286 |
HEB associates with PRC2 and SMAD2/3 to regulate developmental fates |
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| Relations |
| BioSample |
SAMN02980970 |
| SRA |
SRX674278 |
| 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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