Epitope-tagged Asf1 and HIRA cloning

The HA epitope-tagged Asf1a (NCBI GeneID: 25842) and Asf1b (GeneID: 55723) cDNAs as well as the HIRA (GeneID: 7290) cDNA were a gift from Peter Adams (Beatson Institute, Glasgow). The HA-Asf1a and HA-Asf1b were subcloned in the pENTR1A no ccdB plasmid and the HIRA cDNA was cloned into the pENTR4-V5 plasmid. The XPG (GeneID: 2073) cDNA cloned in to the pENTR3C vector with a V5 epitope and a GFP-fusion at its C-terminus (XPG-V5-GFP) was a gift of Ely Kwoh. The XPG-V5 was subcloned into pENTR3C to remove the GFP fusion. The Ubc9 (GeneID: 7329) cDNA was a gift from Claude Gazin (CNRS, UMR217). The ARID4B (GeneID: 51742) cDNA was obtained from the Kazusa cDNA project (clone HH11923, Accession #AB210032). The ARID4B cDNA was cloned in the pENTR4-V5 plasmid. A region of the cDNA encoding the shorter isoform was amplified by RT-PCR from U2OS cells and subcloned into the pCR2.1 TA cloning plasmid (Invitrogen) and sequence verified. The fragment was excised using the Bbv CI/Hind III restriction enzymes and inserted into the Bbv CI + Hind III-digested pENTR4-V5-ARID4B plasmid to generate pENTR4-V5 ARID4B Δchromo.
The shRNA for Asf1a was derived from an siRNA previously used [84] (link). We inserted the following annealed oligonucleotides between the Bgl II/Hind III sites of either pENTR/pTER+ or pENTR/pSUPER+:
For Asf1a:
5′GATCCCGTGAAGAATACGATCAAGTGTGTGCTGTCCACTTGATCGTATTCTTCACTTTTTGGAAA and 5′AGCTTTTCCAAAAAGTGAAGAATACGATCAAGTGGACAGCACACACTTGATCGTATTCTTCACGG, where the underlined sequence is specific for human Asf1a.
For MDC1: 5′GATCCCCCAACATGCAGAGATTGAAATTCAAGAGATTTCAATCTCTGCATGTTGTTTTTGGAAA and 5′AGCTTTTCCAAAAACAACATGCAGAGATTGAAATCTCTTGAATTTCAATCTCTGCATGTTGGGGFor XPG: 5′GATCCCAGAATACATGCGGTGGATTTTCAAGAGAAATCCACCGCATGTATTCTTTTTTGGAAA and AGCTTTTCCAAAAAAGAATACATGCGGTGGATTTCTCTTGAAAATCCACCGCATGTATTCTGG.
For the Asf1a shRNA, we also designed an miRNA-based loop in the shRNA because it was reported to result in better depletion efficiencies [85] (link). To design the Asf1a miRNA, we used the algorithm from Open Biosystems (www.openbiosystems.com). The following oligos were amplified with the Xho I and Eco RI amplification primers and subcloned into the pENTR/pSM2 vectors according to the manufacturer's protocol:
5′TGCTGTTGACAGTGAGCGAGGTCACAAGATTCCACATTAATAGTGAAGCCACAGATGTATTAATGTGGAATCTTGTGACCCTGCCTACTGCCTCGGA5′TGCTGTTGACAGTGAGCGAAGGTAGAATACTTTCATTATTTAGTGAAGCCACAGATGTAAATAATGAAAGTATTCTACCTCTGCCTACTGCCTCGGAwhere the underlined sequences are specific for the human Asf1a.