Sample 102
Homo sapiens
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.
ENA-FIRST-PUBLIC
2020-05-12T04:07:20Z
ENA-LAST-UPDATE
2019-10-23T14:38:21Z
External Id
SAMEA6109411
INSDC center name
Max Planck Institute for Infection Biology, Department of Cellular Microbiology
INSDC first public
2020-05-12T04:07:20Z
INSDC last update
2019-10-23T14:38:21Z
INSDC status
public
Submitter Id
E-MTAB-8459:Sample 102
broker name
ArrayExpress
cell line
PLB-985
common name
human
developmental stage
adult
disease
acute myeloid leukemia
genotype
H1.3 knockout clone1
growth condition
neutrophil differentiation
organism part
blood
sample name
E-MTAB-8459:Sample 102
scientific_name
Homo sapiens