characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2022-08-25T00:13:46Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2022-08-25T00:13:46Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA110654045
characteristics.characteristic: External Id
characteristics.text: WIS
characteristics.characteristic: INSDC center name
characteristics.text: 2022-08-25T00:13:46Z
characteristics.characteristic: INSDC first public
characteristics.text: 2022-08-25T00:13:46Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS12751627
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-12061:Sample 271
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.characteristic: broker name
characteristics.text: OCI-AML3
characteristics.characteristic: cell_line
characteristics.text: mononuclear cell
characteristics.characteristic: cell_type
characteristics.text: human
characteristics.characteristic: common name
characteristics.text: Protocols: Cell line was sub-cultured in RPMI 1640 Medium containing L-Glutamine (Biological Industries, 01-100-1 A) with 10% FBS, streptomycin (20 mg/mL), and penicillin (20 unit/mL) at a density of 3*105 cells/ml, at 37°C in a humidified 5% CO2 incubator. All electroporation reactions were performed using the 16-strip Lonza 4D nucleofector kit. Pre-electroporated cells were washed in PBS and spun down at 350xg for 10 min. A range of 2*105 - 3*105 cells per reaction were re-suspended in 20 ul SF solution (K562), SE solution (OCI-AML3) and added to the RNP complex and 1.5 ul of ASXL1 or SRSF2 single guide. FF-120 and EO-100 electroporation programs were used for K562 and OCI-AML3 respectively. Immediately after electroporation, pre-warmed media were added, and cells were cultured at the same conditions as the pre-electroporation culturing for four days before they were single-cell sorted. CRISPR library: Lyophilized sgRNAs were re-suspended in IDTE buffer (PH 7.5) to a final concentration of 100 uM. RNP complex for each reaction was generated by mixing 1.2 ul sgRNA, 1.7 ul Cas9 protein (IDT), and 2.1 ul PBS followed by incubation for 10 min at 20 degrees. Cells were spun at 1000g for 5 min. Cells pellets were mixed with 50 ul of 50 mM NaOH and heated at 99 u00b0C for 10 min. Then, the reactions were cooled down at room temperature, and 5 ul 1 M Tris PH = 8 was added to each reaction. cells were spun at 1000g for 5 min. Cells pellets were mixed with 50 ul of 50 mM NaOH and heated at 99 u00b0C for 10 min. Then, the reactions were cooled down at room temperature, and 5 ul 1 M Tris PH = 8 was added to each reaction. An amount of 200 ng of genomic DNA from each sample underwent bisulfite conversion with Zymo DNA Methylation Gold Kit (Zymo Research). The converted DNA served as a template for PCR amplification. Dual indexed Illumina Libraries were generated using a two-step PCR procedure. First PCR primer prefix sequences and second PCR primer sequences were used, similar to a previously described method. First PCR was applied to target the regions of interest. The reaction mixture was composed of a PCR-ready mix (using NEBNextu00ae Ultrau2122 II Q5u00ae Master Mix, NEB, M0544L), a cell lysis product, and a final primer concentration of 1uM, for each primer. 2nd PCR was performed using the Illumina sequencing primers, indexes, and adapters, under similar conditions to the 1st PCR, except for the final primer concentration of 0.5uM for each primer, and the number of amplification cycles (n=20)
characteristics.characteristic: description
characteristics.text: leukemia
characteristics.characteristic: disease
characteristics.text: CRISPR/Cas9 double strand break library
characteristics.characteristic: genotype
characteristics.text: Homo sapiens
characteristics.characteristic: organism
characteristics.text: Homo sapiens
characteristics.characteristic: organism
characteristics.text: blood
characteristics.characteristic: organism part
characteristics.text: Homo sapiens
characteristics.characteristic: scientific_name
characteristics.text: Sample 271
characteristics.characteristic: title