Ubp-M knock-down cell lines
Ubp-M knock-down cell lines were generated using a vector-based knockdown strategy as previously described (Wang et al., 2004).
- Transfect vectors expressing the hairpin RNAs as listed below into HeLa cells with Effectene (Qiagen).
- Select transfected cells in the presence of 2 ug/ml puromycin. 3. Amplify selected clones and analyse the efficiency of Ubp-M knockdown as well as its effect on cellular uH2A levels were analyzed as described (Wang et al., 2004).
The DNA sequence encoding the stem hairpin RNA for Ubp-M: 5’-ACCAGTGCTTAGAGAACTATCTCTTGAATAGTTCTCTAAGCACTG
GT-3’ and 5’-ACCAGTGCTTAGAGAACTATTCAAGAGATAGTTCTCTAAGCA
CTGGT-3’.
For transient knockdown, purchase siRNA oligonucleotides against Ubp-M from Invitrogen in a purified, annealed duplex form and transfect into cells as described (Wei et al., 2006). The sequences for these siRNA are as follows:
siRNA1
5’-CCUCCUGUUCUUACUCUUCAUUUAA-3’
5’-UUAAAUGAAGAGUAAGAACAGGAGG-3’ and
siRNA2
5’-CCGGAAAUCUUAGAUUUGGCUCCUU-3’
5’-AAGGAGCCAAAUCUAAGAUUUCCGG-3’
RNAi resistant Ubp-M constructs
- To construct RNAi resistant Ubp-M constructs, introduce a point mutation into targeting siRNA nucleotide acid sequence but without changing the encoded amino acid.
- Perform site-directed mutagenesis as described (Wang et al., 2004).
- Use Primers F1: 5’-AAGGATCCGGAAAGAAACGGACAAAGGGAAAAACTGTTCC-3’ and R1: 5’-TTCTTTTAGGAGCTCCCTGAGTACAGGAGTTTGTGACAA-3’ to amplify the N-terminal portion.
- Use Primers F2: 5’-CAGAACTTGTCACAAACTCCTGTACTCAGGGAGCTCCTAAA-3’ and R2: 5’-GATCTAGATTACAGTATTCTCTCATAAAATAGGAGGTACG-3’ to amplify the C-terminal portion.
- Amplify the full length Ubp-M construct using primers F1 and R2 and inserted into pcDNA3 vector (Invitrogen).
- Verify the mutation by DNA sequencing.
- Perform transfection with Effectene (Qiagen) following the manufacturer’s instruction.
- 72 hrs after transfection, seed and transfect cells with the same plasmid again.
- Collect cells six days after the first transfection and analyze by RT-PCR as described (Wei et al., 2006).
Primers for RT-PCR amplification:
HoxA9: 5’-ACGTGGACTCGTTCCTGCTG-3' and
5’-AGGTTTAATGCCATAAGGCCG-3’
HoxB1: 5’-TCAGGCGGTTGACAGCTATG-3’ and
5’-ATGCTGCGGAGGATATGGC-3’
HoxC5: 5’-TGTGGGAACTATGGATCGGC-3’ and
5’-ACGGGTAAATCTGTGGCGG-3’
HoxD10: 5’-GATTCCTTGATCAGTGCCTGC-3’ and
5’-GCCGAAATGAGTTTGTTGCG-3’
GAPDH: 5’-ACCACAGTCCATGCCATCAC-3’ and
5’-TCCACCACCCTGTTGCTGTA-3’
ChIP and double ChIP
Perform ChIP and double ChIP as described with modifications as the following 5.
Lyse cells in lysis buffer [1% SDS, 10 mM EDTA, 50 mM Tris-HCl (pH 8.0), 0.1 mM PMSF, 1 ug/ml aprotinin, 1 ug/ml leupeptin and 1 ug/ml pepstatin A] and dilute 10 times with dilution buffer [1 % Triton X-100, 150 mM NaCl, 2 mM EDTA, 20 mM Tris-HCl (pH 8.0), 0.1 mM PMSF, 1 ug/ml aprotinin, 1 ug/ml leupeptin and 1 ug/ml pepstatin A] for immunoprecipitation.
Perform PCR reactions with the following primers:
A: 5'-CTTCATTCAGCTTTGGGCACGCTT-3' and
5'-GGCCTGGTTGAAACAAGCGTTGAA-3'
B: 5’-CCCAGAATGCTGAGGCGCTTTAAT-3’ and
5’-ACCACCACTACCACCATGGAAACT-3’
C: 5’-TGGTTCTTTAATGAGCCGGACCAC-3’ and
5’-TGTTGCCACACACGGGAAGATACT-3’
D: 5’-TGTCCTTCTTGGCCCAGTCAGTTT-3’ and
5’-TAAGAAGCGGCCAAGGTGTCTCTA-3’
E: 5’-TAGAGACACCTTGGCCGCTTCTTA-3’ and
5’-CACGGACAACAGCGACATCTACTA-3’
F: 5’-ATGTCCTTTCCCAACAGCTCTCCT-3’ and
5’-AAATATCCAGGGACGGGAACCTCA-3’
G: 5’-TGGTTGGTGCTTGAGTTGGGAAAC-3’ and
5’-TTGCCTGGGTATTGTTGCATTCCC-3’
H: 5’-TCACCGACAGGCAGGTCAAGATTT-3’ and
5’-TGGATGGATGGATGGATGGATGCT-3’
- In the PCR reaction, include one cycle of 5 min at 95°C followed by 33 cycles of 95°C for 30 sec, 68°C for 30 sec, 72°C for 30 sec for regions A, B, C, E, F and G. For D, use an annealing temperatures of 66°C, and for H, 60°C. Perform all PCR reactions within the range of linear amplification.