title: AG12
organism: Homo sapiens
description: Protocols: HEK293T cells were cultured in Dulbecco's modified Eagle Medium (DMEM) (Life Technologies) containing 10% fetal bovine serum (Life Technologies), 2 mM glutamine (Life Technologies), 7mM MEM Non-Essential Amino Acids (Life Technologies), 1mM Sodium Pyruvate (Life Technologies), 10mM HEPES,100 units/ml penicillin streptomycin, and 100 μg/ml streptomycin (Life Technologies). Cells were co-transfected with constructs expressing 5' and 3' sgRNAs and pBABE-Puro in Opti-MEM® Reduced Serum Medium (Life Technologies) using the Lipofectamine® 3000 Reagent (Life Technologies). Cells were co-transfected in 48 well plates with either 0.75 μg of 5'sgRNA, 0.75 μg, 3' sgRNA and 0.5 μg pBABE-Puro or 1.5 μg of empty PHI74 vector and 0.5 μg pBABE-Puro. 48 hours after transfection HEK293T cells were cultured for 14 days in the presence of Puromycin (0.75 μg/ml). 72 hours after transfection cells were split onto 10 cm dishes to ensure clonal growth. 10-14 days after splitting, clones were picked, amplified and tested for CRISPR-CaS11-mediated deletion by PCR. Homozygous clones were further amplified. Clones were harvested on different dates and pellets were frozen; however, all samples were processed simultaneously for DNA and RNA extraction (ATAC-seq and RNA-seq), library generation and sequencing. Total RNA was assessed for quality using an Agilent Tapestation Samples determined to have an RNA Integrity Number (RIN) of 7 or greater were used to generate RNA libraries using Illumina's TruSeq Stranded Total RNA Sample Prep Kit
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2017-06-15T17:01:31Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2017-05-16T18:32:20Z
attributes.tag: External Id
attributes.value: SAMEA104032929
attributes.tag: INSDC center name
attributes.value: unspecified
attributes.tag: INSDC first public
attributes.value: 2017-06-15T17:01:31Z
attributes.tag: INSDC last update
attributes.value: 2017-05-16T18:32:20Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-5714:AG12
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: HEK293T
attributes.tag: cell type
attributes.value: epithelial cell
attributes.tag: common name
attributes.value: human
attributes.tag: genotype
attributes.value: chr8:579137-581436 deletion
attributes.tag: organism part
attributes.value: kidney
attributes.tag: sample name
attributes.value: E-MTAB-5714:AG12
attributes.tag: scientific_name
attributes.value: Homo sapiens