HA-MacroH2A2 inducible MEF cells isolated from HA-MacroH2A2 embryos
chip antibody
HA antibody, Abcam ab9110
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Eluted ChIP materials were PCI (Phenol-Chloroform-Isoamylalcohol) extracted ChIP material was gel purified (100 to 800bp in size) from a 2% TAE agarose gel using MiniElute columns QiaQuick (Qiagen). Gel purified DNA fragments were blunt ended and phosphorylated with an EPICENTRE End-it-Repair kit (1X buffer, 0.25mM dNTPs,1mM ATP, 1ul/50ul reaction of Enzyme mix) for 1hr at RT and cleaned up with Qiagen MiniElute spin columns. Adenosine nucleotide overhangs were added using EPICENTRE exo- Klenow for 45min at RT (with 0.2mM dATP). Illumina genome sequencing adaptors were then ligated using the EPICENTRE Fast-Link ligation kit: 11.5µl A tailed DNA eluted from a MinElute column was mixed with 1.5µl 10X ligation buffer, 0.75µl 10mMATP, 0.5µl Illumina DNA adaptors and 1µl ligase. The reaction was incubated for 1hr at RT and subsequently supplemented with 7.5 µl water, 1µl 10X buffer, 0.5µl 10mM ATP and 1µl ligase, and incubated overnight at 16οC. The ligation reaction was cleaned up with MiniElute columns (with an additional wash step to eliminate all the excess adaptors) and the adaptor ligated fragments were amplified by PCR as follows:0.75 µl of each Illumina genomic DNA sequencing primers, 6µl 10xPfx buffer 1.8µl 10mM dNTPs, 1.2µl 50mM MgSO4 and 1µl Pfx DNA polymerase (Invitrogen) were added to 30µl DNA template in a 100ul reaction. The cycling parameters were: 1. 94οC 2’; 2. 94οC 15’’; 3. 65οC 1’; 4. 68οC 30’’; 5. repeat from step 2 16 times; 6. 68οC 5’. The PCR product (250 to 450bp in size) was gel purified from a 2% TAE agarose gel using the QiaQuick columns (Qiagen). Gel purified fragments were finally precipitated with Sodium acetate and ethanol and pellets were resuspended (25nM final concentration) in TE buffer.