4 protocols
normalization data transformation protocol
Biological replicates were mapped against the human genome using Bowtie and pooled Genomic intervals bound by macroH2A.1 were called using SICER (Window size= 200, Fragment size= 260, Gap size= 600, FDR= 0.01) Supplementary_files_format_and_content: bed; bound intervals
nucleic acid library construction protocol
About 4 million cells were used for immunoprecipitation using anti HA antibodies (Ab9110 from Abcam).The chromatin immunoprecipitation (ChIP) assays were performed according to the Millipore protocol. Cells were fixed using 1% formaldehyde, harvested, resuspended in ChIP lysis buffer (1% SDS, 10mM EDTA, 50mM Tris-HCl, pH 8.1) and sonicated using the Branson Digital Sonifier to generate fragments of 150 to 500 bp. Soluble chromatin was diluted 8 fold in ChIP RIPA buffer (10 mM Tris–HCl, pH 7.5, 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% SDS, 0.1% Na-deoxycholate) and incubated with Dynabeads Protein A (Invitrogen) coupled to specific antibodies against HA (Ab9110 from Abcam). After incubation, the immunocomplexes were washed sequentially with Low Salt Wash Buffer (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCl, pH 8.1, 150mM NaCl), High Salt Wash Buffer (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCl, pH 8.1, 500mM NaCl), LiCl Wash Buffer (0.25M LiCl, 1% NP40, 1% deoxycholate, 1mM EDTA, 10mM Tris-HCl, pH 8.1) and TE. Immunocomplexes were eluted in ChIP elution buffer (1%SDS, 0.1M NaHCO3) and the crosslinking was reverted overnight at 65ºC. Samples were treated with Proteinase K and RNase A and DNA was extracted using the Qiagen PCR purification kit. Libraries for input and immunoprecipitated material were constructed selecting fragments from 200 to 500 bp and processed in the Solexa GAIIx Sample Sequencing (single reads of 36 nt)
sample treatment protocol
Transduced with lentiviral particles pWPI-HA:macroH2A.1
growth protocol
Human keratinocytes were cultured using Epilife (invitrogen)