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Sample GSM1631492 Query DataSets for GSM1631492
Status Public on May 11, 2015
Title INT_Replicate_1
Sample type SRA
 
Source name HEK-293FT
Organism Homo sapiens
Characteristics cell line: HEK-293FT
grna: INT
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).
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.
Extracted molecule total RNA
Extraction protocol RNA: TriZol
Illumina TruSeq
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2500
 
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
 
Submission date Mar 10, 2015
Last update date May 15, 2019
Contact name Scott T Younger
E-mail(s) styounger@cmh.edu
Organization name Children's Mercy Kansas City
Street address 2401 Gillham Road
City Kansas City
State/province MO
ZIP/Postal code 64108
Country USA
 
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
Relations
BioSample SAMN03397543
SRA SRX952180

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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