title: Cntrl#1_3
organism: Homo sapiens
description: Protocols: Human triple negative breast cancer cells Hs578T were cultured in DMEM supplemented with 10% fetal bovine serum in the presence of 1% penicillin-streptomycin. Hs578T cells were transfected with CRISPR Cas9 and HDR plasmids obtained from Santa Cruz Biotech. targeting the human ZEB1 (Santa Cruz Biotech., sc-423302 and sc-423302-HDR) gene. Two days post-transfection, cells were selected with puromycin and single cell colonies were picked and cultured. The obtained clones were then validated using western blotting and the deleted sequences were validated using conventional PCR amplification and sequencing of DNA fragments spanning the sites of guide sequences employed in the CRISPR Cas9 vector system. The TRIzol extraction protocol was used to extract total RNA from both wild type clones and knockout clones using TRIzol reagent from Ambion/Life Technologies and based on the manufacturer's protocol. Total RNA for Ampliseq was extracted with 3 biological replicates and 3 technical replicates as described above. Fifty ng of RNA was reverse transcribed to cDNA using the Ion AmpliSeq™ Transcriptome Human Gene Expression Kit Preparation protocol (Revision A.0, Life Technologies). The acquired cDNA was amplified using Ion AmpliSeq™ Transcriptome Human Gene Expression core panel (Life Technologies) and the primer sequences were then partially digested. Then, DNA adaptors (Ion P1 Adaptor and Ion Xpress™ Barcode Adaptor, Life Technologies) were ligated to the amplicons. Adaptor ligated amplicons were purified using the Agencourt® AMPure® XP reagent (Beckman Coulter) and eluted in amplification mix (Platinum® PCR SuperMix High Fidelity and Library Amplification Primer Mix, Life Technologies) and amplified. Size-selection and purification was conducted using Agencourt® AMPure® XP reagent (Beckman Coulter). The amplicons were quantified using the Fragment AnalyzerTM instrument (Advanced Analytical Technologies, INC.) with DNF-474 High Sensitivity NGS Fragment Analysis Kit (Advanced Analytical Technologies, INC.). Samples were then pooled, six or less per pool, followed by emulsion PCR on either the Ion OneTouch™ 2 System using the Ion PI™ Hi-Q™ OT2 Kit (Life Technologies), or on the Ion Chef™ System using the Ion PI Hi-Q Chef Kit (Life Technologies). The pooled samples were loaded on Ion PI™ v3 chips and sequenced on the Ion Proton™ System using the Ion PI™ Hi-Q Sequencing 200 Kit chemistry (200 bp read length, Life Technologies). Mean read length was 114 bp. Acquired reads were then aligned to the hg19 AmpliSeq Transcriptome ERCC v1 using the Torrent Mapping and Alignment Program (TMAP) with default settings. Differentially expressed genes were called requiring a mean log2-fold change over the replicates of 2 and a q-value < 0.05 (one-sided t-test with False Discovery Rate (FDR) adjustment).
attributes.tag: ENA first public
attributes.value: 2018-04-25
attributes.tag: ENA last update
attributes.value: 2016-11-16
attributes.tag: External Id
attributes.value: SAMEA4552435
attributes.tag: INSDC center alias
attributes.value: Ludwig Cancer Research and Institute of medical biochemistry and microbiology, SciLife lab, Uppsala University, Sweden.
attributes.tag: INSDC center name
attributes.value: Ludwig Cancer Research and Institute of medical biochemistry and microbiology, SciLife lab, Uppsala University, Sweden.
attributes.tag: INSDC first public
attributes.value: 2018-04-25T17:02:54Z
attributes.tag: INSDC last update
attributes.value: 2016-11-16T15:19:35Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-5243:Cntrl#1_3
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: HS578T
attributes.tag: cell type
attributes.value: epithelial cell
attributes.tag: common name
attributes.value: human
attributes.tag: disease
attributes.value: breast carcinoma
attributes.tag: genotype
attributes.value: wild type
attributes.tag: sample name
attributes.value: E-MTAB-5243:Cntrl#1_3