SLX-14481.iPCRtagT003
Homo sapiens
Protocols: Cells were collected at 70-80% confluence. Five 150 mm dishes were used for each pulldown. Cells were trypsinized, washed with phosphate buffer saline solution, and fixed twice with 1% formaldehyde. HeLa cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum at 37 degrees Celsius in 5% CO2. Nuclei were extracted and cross-linked with 3% formaldehyde for 30 minutes at room temperature. Nuclei were washed 5 times after quenching with 0.125 M glycine. Sonication was performed using a Covaris S220. Extracts were collected and hybridised to capture oligonucleotides (The antisense pool was comprised of 4 separate oligonucleotides with sequences derived from the antisense strand of LINC00899, the antisense pool, derived from the sense strand of LINC00899, was a mix of 2 separate oligonucleotides, which were the direct reverse complement sequences of two of the oligos in the antisense pool) at a final concentration of 25 uM, overnight at room temperature. A portion of the nuclear extract incubated with either the sense or antisense capture oligos was removed prior to pulldown to serve as input controls. Streptavidin C1 beads were used to capture complexes overnight at room temperature. Bound material was washed 5 times and RNAse H was added for 30 minutes at room temperature to elute RNA-chromatin complexes. Eluates were reverse-cross-linked and treated with Proteinase K (55 degrees Celsius for 1 hour, then 65 degrees Celsius for 30 minutes). DNA was purified using phenol-chloroform extraction and ethanol precipitation. Final CHART DNA was eluted in 12.5 uL of Tris-EDTA buffer. 10 uL from this reaction was used for sonication using the Covaris LE220 to obtain fragment lengths below 500 bp. CHART DNA was used for library preparation using the ThruPLEX DNA-seq library preparation protocol (Rubicon Genomics). For inputs, 6 PCR cycles were used, while for pulldown eluates, 8 PCR cycles were used. Library fragment sizes were performed using a 2100 Bioanalyzer and libraries quantified by the KAPA quantification kit.
ENA-FIRST-PUBLIC
2019-03-29T17:02:07Z
ENA-LAST-UPDATE
2018-11-27T14:40:38Z
External Id
SAMEA5137571
INSDC center name
CRUK Cambridge Institute
INSDC first public
2019-03-29T17:02:07Z
INSDC last update
2018-11-27T14:40:38Z
INSDC status
public
Submitter Id
E-MTAB-7418:SLX-14481.iPCRtagT003
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-7418:SLX-14481.iPCRtagT003
scientific_name
Homo sapiens