Homo sapiens
Protocols: HeLa cells were washed in cold PBS and harvested by scraping into cold PBS followed by centrifugation at 300 x g. DNA from up to 10^6 cells was extracted using a Quick-DNA™ kit (Zymo Research) according to the manufacturer's instructions. Genomic lysis buffer was supplemented with TEMPO (20 mM) to minimise the introduction of oxidative damage and DNA was eluted in Tris-HCl buffer (pH 7.4, 10 mM). Detailed protocol is available in methods section. Briefly, purified DNA was sonicated using a Covaris M220 system, to an average length of 450 bp. Sonicated DNA was treated with HIPS probe 1 (10 mM, 2 hr, RT) and biotinylated using the CuAAC reaction and biotin-PEG3-azide (2 hr, 37 °C). A custom P7 adapter was introduced onto DNA by ligation using a NEBNext Ultra II kit. Labelled DNA was isolated using magnetic streptavidin beads and unbound DNA was removed by washing. The enriched DNA was denatured on the beads using NaOH solution (RT) and then eluted using NaOH solution (70 °C). Eluted DNA was purified once again on magnetic streptavidin beads to remove any non-specific elution. A single primer extension was performed on eluted ssDNA, then a custom P5 adapter was introduced by ligation. Libraries were amplified by PCR.
ENA-FIRST-PUBLIC
2019-06-12T17:06:27Z
ENA-LAST-UPDATE
2018-09-04T14:35:11Z
External Id
SAMEA4883116
INSDC center name
Department of Chemistry University of Cambridge
INSDC first public
2019-06-12T17:06:27Z
INSDC last update
2018-09-04T14:35:11Z
INSDC status
public
Submitter Id
E-MTAB-7152:APE1-siRNA_snAP4
broker name
ArrayExpress
cell line
HeLa
cell type
epithelial cell
common name
human
disease
cervical adenocarcinoma
genotype
wild type genotype
organism part
uterine cervix
sample name
E-MTAB-7152:APE1-siRNA_snAP4
scientific_name
Homo sapiens