NextSeq 500 sequencing; ZXDA/B and CENP-A/B Chromatin immunoprecipitation sequencing (ChIP-seq) in human colon carcinoma cells.
ZXDA/B and CENP-A/B Chromatin immunoprecipitation sequencing (ChIP-seq) in human colon carcinoma cells.
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 g/l) overnight at 65 C, followed by one hour incubation with Proteinase K (Santa Cruz;0.3 g/l) 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.