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Sample GSM1631262 Query DataSets for GSM1631262
Status Public on Mar 11, 2015
Title dCas9_rep1
Sample type SRA
Source name Cultured HEK293T cells
Organism Homo sapiens
Characteristics cell line: HEK293T
transfected gene: dcas9
guide rna at 4 sites: IL1RN
Treatment protocol HEK293T cells were transfected with Lipofectamine 2000 (Invitrogen) according to manufacturer’s instructions. Transfection efficiencies were routinely higher than 80% as determined by fluorescence microscopy following delivery of a control eGFP expression plasmid. Cas9 expression plasmids were transfected at a mass ratio of 3:1 to the total amount of gRNA expression plasmids as in main text.
Growth protocol HEK293T cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cel Culture Facilities and were maintained in DMEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C with 5% CO2.
Extracted molecule total RNA
Extraction protocol Between 0.5-1 million cells were washed with PBS and then lysed using Qiagen RLT buffer. Total RNA was extracted using Qiagen RNeasy mini-columns with 1% β-mercaptoethanol in RLT buffer and a 15 min on column DNase digestion. mRNA was prepared using the Wafergen automated sample prep system with the Prep-x mRNA kit with 1 microgram of total RNA.
Libraries were prepared using the Apollo 324 liquid handling platform, as per manufacturer’s instruction using the prep-mRNAseq kit.
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2500
Description CV41_CGATGT
Data processing Base-calling was performed on instrument using CASAVA software
Reads were aligned to the hg19 version of the human genome using bowtie2-align version 2.1.0, and the -p 32 --end-to-end --very-sensitive t" alignment options
Reads were converted from .sam to .bam files using samtools view version 0.1.19-44428cd with the "-bS" option
Bam files were sorted using Samtools sort version 0.1.19-44428cd with the options "32 -m 2G"
Bam files were indexed using Samtools Index version 0.1.19-44428cd
Duplicate reads were removed using samtools rmdup version 0.1.19-44428cd  usint the "-s option"
Bam files were re-indexed after duplicates were removed using Samtools Index version 0.1.19-44428cd
Reads were counted using samtools idxstats version 0.1.19-44428cd
Differential expression was computed using R version 3.1.2 using the DEseq2 package version 1.6.2 with default settings
Genome_build: RefSeq transcripts from hg19
Supplementary_files_format_and_content: Counts of the number of reads aligned to each RefSeq transcript.
Submission date Mar 10, 2015
Last update date May 15, 2019
Contact name Christopher Vockley
Organization name Duke University
Street address 101 science drive rm 2193
City Durham
State/province NC
ZIP/Postal code 27708
Country USA
Platform ID GPL16791
Series (1)
GSE66742 Epigenome Editing by a CRISPR/Cas9-Based Acetyltransferase Activates Genes from Promoters and Enhancers
BioSample SAMN03397426
SRA SRX950686

Supplementary file Size Download File type/resource
GSM1631262_dcas9_IL1RN_1.counts.txt.gz 180.6 Kb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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