characteristics.text: heterogeneous dCas9-KRAB
characteristics.characteristic: Characteristics [genotype
characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2018-03-22T17:02:19Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2018-03-22T09:22:05Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA104711599
characteristics.characteristic: External Id
characteristics.text: Cancer Research UK Cambridge Institute
characteristics.characteristic: INSDC center name
characteristics.text: 2018-03-22T17:02:19Z
characteristics.characteristic: INSDC first public
characteristics.text: 2018-03-22T09:22:05Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS2312982
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-53081521706845:do17890
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: HeLa
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0001185
characteristics.characteristic: cell line
characteristics.text: human
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_9606
characteristics.characteristic: common name
characteristics.text: Protocols: HeLa cells were obtained from American Type Culture Collection. To generate a heterogeneous population of cells expressing dCas9-KRAB, HeLa cells were transduced with a lentiviral vector containing the pHR-SFFV-dCAS9-BFP-KRAB vector along with polybrene (5 ug/ml, Sigma), incubated for 72 hours with medium replacement after 24 hours, and sorted for the BFP-expressing cells using a BD FACSAria III cell sorter. To generate single-cell clones expressing dCas9-KRAB, BFP-sorted single cells were plated on 96-well plates and grown for two weeks until colonies were obtained. Expression of dCas9-KRAB in each colony was verified by Western blot. HeLa cells were maintained in Dulbecco's modified Eagle's medium (Sigma Aldrich, D6429) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific) and cultured at 37 degrees Celsius with with 5% CO2. For RNAi and LNA, HeLa cells were transfected with Lipofectamine RNAiMax reagent (Thermo Fischer Scientific) following the manufacturer's instructions, using siRNAs (Thermo Fischer Scientifc) and LNA Gapmers (Exiqon) at a final concentration of 50 nM and 25nm, respectively. All experiments were done 48 hr after transfection. For CRISPRi, dCAS9-KRAB transduced cells were plated on a 12-well plate and infected with lentivirus containing the appropriate sgRNA vector. The virus was diluted with HeLa media (1:1 dilution) and cationic polymer polybrene was added to facilitate viral transduction (5 ug/ml, Sigma). After 24 hr incubation, supernatant was removed and fresh media was added for 48 hr before RNA collection. Samples with the same batch number were treated at roughly the same time (within the same month), while the experiment number is nested within batch and refers to cells treated on the same day. The QuantiTect Reverse Transcription Kit (QIAGEN) was used for cDNA synthesis including an additional step to eliminate genomic DNA contamination. RNA (1 ug) was extracted with the RNeasy Kit (QIAGEN) and treated with DNase I following the manufacturer's instructions. RNA quality was assessed using a Total RNA Nano chip with a 2100 Bioanalyzer instrument (Agilent). RNA-seq libraries were prepared from HeLA cells using TruSeq Stranded Total RNA Kit with Ribo-Zero Gold (Illumina, RS-122-2303). Library quality was assessed using a DNA1000 chip with a 2100 Bioanalyzer instrument (Agilent).
characteristics.characteristic: description
characteristics.text: CRISPRi
characteristics.characteristic: loss of function method
characteristics.text: Homo sapiens
characteristics.characteristic: organism
characteristics.text: Homo sapiens
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_9606
characteristics.characteristic: organism
characteristics.text: Homo sapiens
characteristics.characteristic: scientific_name
characteristics.text: do17890
characteristics.characteristic: title