Plasmid Construction and Expression of RTTN

The human RTTN cDNA (accession number, BC156291) was purchased from the IMAGE clone consortium (IMAGE clone No. 100061722) and subcloned into pEGFP-N2 (BD Biosciences Clontech) or pFlag-CMV2 (Sigma-Aldrich). To generate the GST-fusion constructs, the cDNAs encoding various potion of RTTN were fused in-frame to the GST-encoding sequence in the pGEX4T vector (GE Healthcare). The RTTN mutant constructs were generated by site-directed mutagenesis using a QuikChange kit (Stratagene). The expression constructs encoding various GFP-tagged RTTN mutant proteins were generated using pcDNA4/To/myc-His-A (Invitrogen) or pLVX-Tight-Puro (BD Biosciences Clontech) vectors. Sequencing was used to confirm all generated plasmids. The cDNAs encoding full-length CP110, H2B and STIL were obtained by RT-PCR using total RNAs from human HEK293T cells and subcloned in-flame into a pEGFP-C1 (BD Biosciences Clontech) or a pLVX-Tight-Puro (BD Biosciences Clontech) vector. The cDNA constructs for GFP-tagged full-length STIL, SAS-6, and CPAP and various Flag-tagged STIL truncated mutants were from ref. ^{8 (link)}. The cDNA constructs containing various CEP295 fragments were from ref. ^{19 (link)}.