Homo sapiens
Protocols: Cells were lysed 48 h after transfection in cold lysis buffer (10mM Tris-HCL, pH 7.4, 10mMNaCl, 3mM MgCl2 and 0.1% Igepal). DNA was purified using Qiagen mini elute kit and using Ampure XP beads following the manufacturer's instructions. Nuclei was concentrated by centrifugation at 500 g for 10 minutes at 4°C. The nuclei pellet was resuspended in nuclease free water, diluted 1:10 and quantified. The equivalent volume for 50,000 nuclei was made up to 22.5 ul with nuclease free water. Nuclei were incubated for 30 minutes at 37°C, 300 rpm, with 25 ul of 2x TD buffer and 2.5 ul tagmentation ezyme (Illumina transposase FC-121-1030). Library fragments were amplified using standard PCR and Nextera primers (Illumina) by adding 25 ul of 2x NEBnext master mix, 2.5 ul forward primer, 2.5 ul reverse primer and 20 ul transposased DNA. PCR was run for 5 cycles, 72°C, 5 minutes; 98°C 30 seconds; cycle of 98°C 10 seconds, 63°C 30 seconds, 72°C 1 minute, paused at 4°C. To determine the total number of cycles needed for the amplified library, 5 ul of the PCR mix were taken and added to a new mix containing 5 ul 2X master mix, 1 ul of each primer, 0.6 ul of 10X SYBR green and 2.4 ul H2O. In a qPCR machine, 20 cycles were run, the total number of cycles for the ATAC library was chosen according to qPCR cycle that gave 1/3 of saturation.
ENA-FIRST-PUBLIC
2023-06-01
ENA-LAST-UPDATE
2023-06-01
External Id
SAMEA111468771
INSDC center alias
Faculty of Biology, Medicine and Health, University of Manchester
INSDC center name
Faculty of Biology, Medicine and Health, University of Manchester
INSDC first public
2023-06-01T00:15:25Z
INSDC last update
2023-06-01T00:15:25Z
INSDC status
public
Submitter Id
E-MTAB-12294:ATACseq U2OS
broker name
ArrayExpress
cell_line
U2OS
common name
human
developmental stage
juvenile stage
disease
osteosarcoma
genotype
wild type genotype
isolate
not applicable
organism part
tibia
sample name
E-MTAB-12294:ATACseq U2OS
scientific_name
Homo sapiens