|Public on Mar 11, 2015
|Cultured HEK293T cells
|cell line: HEK293T
transfected gene: dCas9-p300 D1399Y
guide rna at 4 sites: IL1RN
|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.
|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.
|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.
|Illumina HiSeq 2500
|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.
|Mar 10, 2015
|Last update date
|May 15, 2019
|101 science drive rm 2193
|Epigenome Editing by a CRISPR/Cas9-Based Acetyltransferase Activates Genes from Promoters and Enhancers