Protocols: The transfected cells were selected with puromycin and sorted to get single-cell clones. For ChIP, growing cells were fixed with 1% formaldehyde for 10 min. The reaction was stopped using 125mM glycine, cells were washed twice with PBS and collected. To generate FBXO38 knockout cell lines, the LentiCRISPR (pXPR_001; Addgene, #49535) containing both hSpCas9 and sgRNAs (targeting the sequences AAGCTGTATGACCGTATGTGTGG and GATGCATGTTTTCCGGTGAATGG) were transfected into the previously subcloned HCT116 cells. The cells were lysed in a sonication buffer (50mM Tris pH 7.5, 140mM NaCl, 1mM EDTA pH 8, 0.1% Triton X-100, 0.1% SDS, protease inhibitor cocktail), incubated for 10 min on ice, centrifuged and the pellets were resuspended in an identical buffer. The subsequent sonication was performed for 10 min to generate a fragment size distribution of 100-800 bp (Covaris ME220 Focused-Ultrasonicator, Peak-Power: 75 W, Duty factor: 15 %, Cycle/Burst 1000, water temperature: 6 °C). The sonicated chromatin was precleared by centrifugation, diluted with an IP buffer (as described above) and immunoprecipitated with the corresponding antibodies (ZXDA/B (Atlas antibodies #HPA043789), CENPA (Cell signaling #2186), CENPB (Santa Cruz #sc-376283), control IgG (Cell Signaling #2729)) overnight at 4 °C, followed by incubation with Dynabeads-Protein G (ThermoFisher Scientific). Precipitated DNA/protein complexes were eluted from stringently washed beads using buffer containing 1% SDS and 100mM NaHCO3. Reverse cross-linking of eluates were performed in presence of 200mM NaCl and RnaseA (ThermoFisher Scientific; 0.15 µg/µl) overnight at 65 °C, followed by one hour incubation with Proteinase K (Santa Cruz;0.3 µg/µl) and subsequent purification of DNA with QIAquick PCR Purification Kit (Qiagen). Sequencing libraries were prepared with NEBNext Ultra II DNA Library Prep Kit for Illumina.
ENA first public
2022-06-15
ENA last update
2022-06-15
External Id
SAMEA11953584
INSDC center alias
Laboratory of Functional Genomics and Bioinformatics Institute of Molecular Genetics of the ASCR, v. v. i.
INSDC center name
Laboratory of Functional Genomics and Bioinformatics Institute of Molecular Genetics of the ASCR, v. v. i.
INSDC first public
2022-06-15T12:17:13Z
INSDC last update
2022-06-15T12:17:13Z
INSDC status
public
Submitter Id
E-MTAB-11272:9_WT_input
batch
A
broker name
ArrayExpress
cell line
HCT116
common name
human
developmental stage
adult
disease
colon carcinoma
genotype
wild type genotype
organism part
colon
sample name
E-MTAB-11272:9_WT_input
sex
male