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| Status |
Public on May 11, 2015 |
| Title |
sgRNA_Replicate_1 |
| Sample type |
SRA |
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| Source name |
HEK-293FT
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK-293FT
grna: sgRNA
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| Treatment protocol |
Transfection mixes comprised 59 ng of dCas9~VP, 11.6 ng of U6-driven sgRNA variant plasmid and 679.4 ng pNEB193 carrier plasmid per well, with 2.25 uL Lipofectamine 2000 (Life Technologies), in 18 uL final volume. Three days later, whole cell RNA was isolated by extraction with Trizol Reagent (Life Technologies), following the manufacturer's protocols. RNA was precipitated with isopropanol using GlycoBlue (Life Technologies) as a carrier, and subsequently purified using RNEasy spin columns (QIAgen), following the manufacturer's "RNA Cleanup" protocol, with on-column DNase treatment. mRNA seq libraries were prepared using the TruSeq RNA sample preparation kit, v2 (Illumina).
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| Growth protocol |
125,000 HEK293FT cells were plated per well of gelatinized 12 well plates, in 0.6 mL high glucose Dulbecco’s modified Eagle’s medium (DMEM, Gibco), supplemented with 10% FBS, 1x penicillin/streptomycin and 2 mM L–Glutamine (Gibco). Cells were grown at 37°C and 5% CO2 in a humidified incubator. Cells were transfected one day later with plasmids expression dCas9 and modified sgRNA plasmid, when appropriate, using Lipofectamine 2000 (Life Technologies), following the manufacturer's protocol. Whole cell RNA was harvested three days later.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA: TriZol
Illumina TruSeq
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
RNA-Seq Read Alignment: TopHat 2.0.13 with default options using UCSC known genes as reference transcriptome
RNA-Seq Differential Expression: Cuffdiff 2.2.1 with default options using UCSC known genes as reference transcriptome
Genome_build: hg19 (modified to incorporate sequences for Gaussia and Cypridina luciferase)
Supplementary_files_format_and_content: TXT files of expression matrices with FPKM values for each sample
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| Submission date |
Mar 10, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Scott T Younger |
| E-mail(s) |
styounger@cmh.edu
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| Organization name |
Children's Mercy Kansas City
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| Street address |
2401 Gillham Road
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| City |
Kansas City |
| State/province |
MO |
| ZIP/Postal code |
64108 |
| Country |
USA |
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| Platform ID |
GPL16791 |
| Series (2) |
| GSE66755 |
CRISPR Display: A modular method for locus-specific targeting of long noncoding RNAs and synthetic RNA devices in vivo [RNA-Seq] |
| GSE66756 |
CRISPR Display: A modular method for locus-specific targeting of long noncoding RNAs and synthetic RNA devices in vivo |
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| Relations |
| BioSample |
SAMN03397541 |
| SRA |
SRX952184 |
| 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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