characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2023-10-29T00:20:23Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2023-10-29T00:20:23Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA10563808
characteristics.characteristic: External Id
characteristics.text: Department of Microbiology, Tumor and Cell Biology - Karolinska Institute Science For Life Laboratory, Stockholm
characteristics.characteristic: INSDC center name
characteristics.text: 2023-10-29T00:20:23Z
characteristics.characteristic: INSDC first public
characteristics.text: 2023-10-29T00:20:23Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS8215782
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-11111:YesCas_replicate1_High_mCherry
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.characteristic: broker name
characteristics.text: RKO
characteristics.characteristic: cell_line
characteristics.text: epithelial cell of large intestine
characteristics.characteristic: cell_type
characteristics.text: human
characteristics.characteristic: common name
characteristics.text: Protocols: 50M RKO cells were seeded in 10 flasks (T175) with 5M cells each. Cells were transduced with mCherry-opools-library virus at low MOI - 0.3 using 2 µg/ml polybrene. 48h later after viral transduction cells were treated with puromycin at a concentration of 2ug/ml. After 48h of puromycin selection, cells were split out and expanded for next 48h in selection until FACS sort. Approximately, 22.76 M mCherry-positive cells were FACs (Cell Sorter SH800S, Sony Biotechnology) sorted and expanded for second lentiviral Cas13d-GFP (pXR001:EF1a-CasRx-2A-EGFP) addgene#109049 transduction. For transduction with Cas13d -GFP virus at high MOI, 50M cells were seeded for Replicate 1 and Replicate 2, respectively. 72 h post-transduction, Replicate 1 and 2 were FACS sorted for top 10% high-mCherry and bottom 10% low-mCherry expression of GFP+mCherry positive cells. Non-transduced Cas13d-GFP cells with only mCherry-opools-Library served as control for both the replicates and top 10% high-mCherry and bottom 10% low-mCherry expressing cells were also sorted to serve as control. Genomic DNA was isolated using the QIAamp DNA Blood Mini kit (Qiagen 51106) guide and UMI sequences were amplified by PCR as described in Schmierer et al 2017
characteristics.characteristic: description
characteristics.text: colon carcinoma
characteristics.characteristic: disease
characteristics.text: mCherry-opools-library; Cas13d-GFP
characteristics.characteristic: genotype
characteristics.text: Homo sapiens
characteristics.characteristic: organism
characteristics.text: Homo sapiens
characteristics.characteristic: organism
characteristics.text: colon
characteristics.characteristic: organism part
characteristics.text: mCherry high
characteristics.characteristic: phenotype
characteristics.text: Homo sapiens
characteristics.characteristic: scientific_name
characteristics.text: YesCas_replicate1_High_mCherry
characteristics.characteristic: title