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| Status |
Public on Jun 01, 2013 |
| Title |
mESC_R2D2_Gapmer_2 |
| Sample type |
SRA |
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| Source name |
Mouse Embryonic Stem Cells
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| Organism |
Mus musculus |
| Characteristics |
knockdown chemistry: Gapmer
knockdown target: R2D2 repeat element of linc-FIRRE
cell type: Embryonic Stem Cells
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| Treatment protocol |
siRNAs were transfected using Lipofectamine RNAiMax (5 μl/well) at a final oligo concentration of 75nM; Gapmer oligos were designed with 2-O methyoxyethyl and transfected with Lipofectamine 2000 at a final concentration of 75nM.
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| Growth protocol |
HeLa cells were grown in DMEM + 10% FBS + 1% Pen-strep + 1% L-glutamine at 37ºC at 5% CO2; Mouse ES cells were grown in previously gelatinized (0.2%) dishes with media containing 125 mL DMEM/F12 (Invitrogen), 83.5 μL BSA fraction V (50 μg/ml relative to DMEM) (Invitrogen, 15260-037, 75mg/ml), 125 mL Neurobasal medium (Invitrogen, 21103-049), 625 μL of the Ndiff Neuro2 (200X, relative to Neurobasal medium) (Millipore, SCM012), 2.5 mL B27 minus vitamin A (50X, relative to Neurobasal medium) (Invitrogen, 12587-010), 2 μL beta-mercaptoethanol (for 250 mL medium), 1 μM PD0325901 (Stemgent, 04-0006), 3 μM CHIR99021 (Stemgent 04-0004), 25 μL LIF ESGRO (stock is 107 units LIF/mL from Chemicon, ESG1106), 1% pen-strep (Invitrogen, 15140-163), 1% Non-Essential Amino Acids (Invitrogen, 11140-076), 1% L-glutamine (Invitrogen, 25030-164). The plating density of mES cells was chosen to be 30,000-50,000/cm2.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
1ml of Trizol and 200μl of chloroform were added and the mix was centrifuged at 4°C at 13,000 rpm for 15 minutes. The aqueous layer was cleaned and DNase treated on RNeasy Mini columns.
Illumina TruSeq RNA
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
RNA-Seq of polyA-selected RNA from mouse embryonic stem cells transfected with an RNA/DNA Gapmer targeting the R2D2 repeat region of linc-FIRRE replicate 2
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| Data processing |
Basecalls were performed using CASAVA 1.8
Reads were aligned using Tophat2 with default options
Expression quantification and differential analysis was done using Cuffdiff2 using UCSC reference transcriptome .gtf files (mouse or human) and Tophat2 aligned reads as input and default options.
Cuffdiff output files were processed using cummeRbund for indexing, exploration, and visualization of results.
Genome_build: mm9
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| Submission date |
Mar 14, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Loyal A Goff |
| E-mail(s) |
loyalgoff@jhmi.edu
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| Organization name |
Johns Hopkins University
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| Department |
Institute of Genetic Medicine
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| Lab |
Goff
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| Street address |
733 N. Broadway Avenue
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| City |
Baltimore |
| State/province |
MD |
| ZIP/Postal code |
21205 |
| Country |
USA |
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| Platform ID |
GPL13112 |
| Series (1) |
| GSE45157 |
Trans-chromosomal regulation by a novel lincRNA required for adipogenesis that escapes X-chromosome inactivation |
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| Relations |
| Reanalyzed by |
GSE80797 |
| SRA |
SRX249072 |
| BioSample |
SAMN01977943 |
| 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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