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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jun 30, 2018 |
| Title |
ES cells RC ASO + siNcl replicate3 |
| 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 7 hours. Then, ES cells were transfected with 100 pmol per well of siRNA according to standard Lipofectamine 2000 protocol. Media was changed the next day and cells harvested after 2 days without sorting 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 4000 |
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| Description |
RCN3
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| Data processing |
Reads were quality and adaptor trimmed using trim_galore v0.4.0, 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 Rsubread package feature-counts v1.22.3, parameters: -t exon -T 4 -s 2 -g gene_id
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_ASOexpt2_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) |
[email protected]
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| Organization name |
MRC Laboratory of Medical Sciences
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| Department |
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 |
GPL21103 |
| Series (2) |
| GSE100938 |
Global analysis of gene expression changes following LINE1 inhibition [II] |
| GSE100939 |
Global analysis of gene expression changes following LINE1 inhibition |
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| Relations |
| BioSample |
SAMN07333507 |
| SRA |
SRX2990584 |
| 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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