ko_1gy_d1_live_n2
Homo sapiens
Protocols: 500,000 HAP1 cells were seeded in 6 well plates and incubated for 24 hours. Untreated live cell samples were harvested and snap frozen and treated samples were irradiated with 1Gy IR. Every 24 hours following the IR treatment, live and dead cell samples were taken and snap frozen. Live cells were harvested by washing in PBS, trypsinising and resuspending in media, pelleting at 300g for 5 minutes, washing the cell pellet by resuspending in PBS, re-pelleting and snap-freezing. Dead cells were harvested by transferring media from the well to a 15mL conical tube, pelleting at 100g for 10 minutes, aspirating supernatant and snap freezing. DNA was extracted by resuspending frozen pellets in cytoplasmic lysis buffer (50 mM HEPES pH 7.9, 10 mM KCl2, 1.5 mM MgCl2, 0.34 M sucrose, 0.5% triton, 10% glycerol, 1 mM DTT) + 0.1mg/mL RNase A and incubating for 10 minutes at room temperature. Proteinase K and SDS were added to a final concentration of 1mg/mL and 1% respectively, and extracts were incubated at 60°C for 60 minutes with 500rpm agitation. 1μL of glycogen (Invitrogen 10814010), sodium acetate pH 5.2 to a final concentration of 300mM and 2.5 volumes of 100% ethanol were added to the samples to precipitate the DNA overnight at -20°C. DNA was pelleted at 20,000g for 30 minutes, pellets were washed once in 80% ethanol and re-pelleted before being left to air-dry. DNA was resuspended in nuclease free water and quantified via NanoDrop.  Genomic DNA was fragmented by sonication with a Diagenode Bioruptor Pico Plus for 9 cycles of 30 seconds on / 30 seconds off. Libraries were generated with 200ng of DNA using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB E7645L) using the manufacturers recommended guidelines and 3 PCR cycles.
ENA first public
2026-03-31
INSDC center name
Cancer Research UK/CI, University of Cambridge
INSDC status
public
Submitter Id
E-MTAB-15023:ko_1gy_d1_live_n2
broker name
ArrayExpress
cell condition
live
cell line
HAP-1
cell type
white blood cell
collection date
not collected
common name
human
developmental stage
adult
disease
acute myeloid leukemia
genotype
PTPN12 KO
geographic location (country and/or sea)
not collected
isolate
not applicable
organism part
bone marrow
replicate
2
sample name
E-MTAB-15023:ko_1gy_d1_live_n2
scientific_name
Homo sapiens