title: Sample 6
organism: Homo sapiens
description: Protocols: PLB-985 at day 0, day 3 and day 7 of differentiation (see growth protocol) were collected, total RNA was extracted by lysis in well. PLB-985 were cultured in RPMI (RPMI 1640 medium, gibco) supplemented with 10 % FCS, L-glutamine and antibiotics (25030-024 and 15140-122, gibco), cells were passaged every 3-4 days. For differentiation, cells were counted at d0 and 0.4x106 cells/ml were seeded in differentiation medium (RPMI supplemented with 2.5 % FCS, 0.5% dimethylformamide (DMF, D4551, Sigma), L-glutamine and antibiotics), usually in 6 well plates in a total volume of 3ml. At d4 of differentiation, 2ml of differentiation medium were added per 6 well. PLB-985 were transduced with lentiviral particles containing the sgRNA of interest cloned into lentiCRISPRv2, cells were selected with puromycin (2.5 g/ml) and, if single clones were analyzed, seeded at a density of 0.8 cells/well in 96 wells for limited dilution cloning. Clones were sequenced using the OutKnocker protocol and software24 (Supplementary Table 3). Clones with out of frame indels leading to efficient gene disruption were used for subsequent experiments. Total RNA of PLB-985 at various time points was isolated using RNeasy kit (Qiagen) according to manual. A stranded RNA seq library was constructed using NEB-Next Ultra RNA directional kit with Poly-A tail enrichment.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2020-05-12T04:07:20Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2019-10-23T14:38:22Z
attributes.tag: External Id
attributes.value: SAMEA6109430
attributes.tag: INSDC center name
attributes.value: Max Planck Institute for Infection Biology, Department of Cellular Microbiology
attributes.tag: INSDC first public
attributes.value: 2020-05-12T04:07:20Z
attributes.tag: INSDC last update
attributes.value: 2019-10-23T14:38:22Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-8459:Sample 6
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: PLB-985
attributes.tag: common name
attributes.value: human
attributes.tag: developmental stage
attributes.value: adult
attributes.tag: disease
attributes.value: acute myeloid leukemia
attributes.tag: genotype
attributes.value: H1.4 knockout clone1
attributes.tag: growth condition
attributes.value: neutrophil differentiation
attributes.tag: organism part
attributes.value: blood
attributes.tag: sample name
attributes.value: E-MTAB-8459:Sample 6
attributes.tag: scientific_name
attributes.value: Homo sapiens