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| Status |
Public on Jun 30, 2018 |
| Title |
ES cells RC ASO replicate2 |
| Sample type |
SRA |
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| Source name |
Mouse E14 ES cells
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| Organism |
Mus musculus |
| Characteristics |
cell line: E14Tg2A
cell type: ES cells
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| Treatment protocol |
ASOs were introduced into cells by nucleofection, utilizing an Amaxa Nucleofector 2b device and ES nucleofection kit (Lonza), according to the manufacturer’s instructions. 4-5 million cells were used per nucleofection together with 5 nmol of the indicated ASO. Cells were plated in ES media immediately following electroporation and left to recover for 48 hours. 300,000 ES cells were purified by Lissamine fluorescence by flow cytometry after 2 days and used for RNA extraction.
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| Growth protocol |
E14 ES cells were cultured in DMEM GlutaMAX with Na Pyruvate (Thermo Fisher Scientific), 15% FBS (Atlanta Biologicals), 0.1 mM Non-essential amino acids, 50 U/ml Penicillin/Streptomycin (UCSF Cell Culture Facility), 0.1 mM EmbryoMax 2-Mercaptoethanol (Millipore) and 2000 U/ml ESGRO supplement (LIF, Millipore).
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using QiaGen Mini purifcation kit, according to standard protocols, including on-column DNAse I digestion
Libraries were generated using the NEBnext Ultra Directional mRNA-seq kit using 1ug total DNAse-1 treated RNA per sample, using NEBNex index primers
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
RC-02
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| Data processing |
Reads were quality and adaptor trimmed using UC Davis tools Sickle (v7667f147e6) and Scythe (vc128b19), and standard parameters
Trimmed reads were aligned to mm9 (Thermo Fisher Scientific) using tophat2 v2.0.9, using standard parameters. Setting g-1 was used for aligning repeats to one location in the genome
Bam files were used to generate count data using htseq-count v0.6.1p1, parameters: -f bam -s reverse -t exon -i gene_id -m union
Count data was analyzed in R v3.3.0, using Bioconductor v3.3, Deseq2 v.1.12.3
Genome_build: mm9
Supplementary_files_format_and_content: Percharde_ASOexpt1_raw-reads.xlsx, excel spreadsheet file
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| Submission date |
Jul 07, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Michelle Percharde |
| E-mail(s) |
m.percharde@lms.mrc.ac.uk
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| Organization name |
Imperial College London
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| Street address |
Du Cane Road
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| City |
London |
| ZIP/Postal code |
W12 0NN |
| Country |
United Kingdom |
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| Platform ID |
GPL17021 |
| Series (2) |
| GSE100937 |
Global analysis of gene expression changes following LINE1 inhibition [I] |
| GSE100939 |
Global analysis of gene expression changes following LINE1 inhibition |
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| Relations |
| BioSample |
SAMN07333505 |
| SRA |
SRX2990565 |
| 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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