I'm trying to do CHIP-qPCR on various cells lines on both histone markers and TF. The problem I've encountered is both high background in non-binding intergenic regions and also high binding in GOI-KO cells using the GOI ab.
Brief protocol is as follows:
Fixation on dish of ~15M cells (10cm dish) with 1% PFA 10min, followed by 0.25M quenching. Cells were then lysed in 0.7%SDS lysis buffer for 10min and sonicated with probe sonicator. Small portion was reverse crosslinked overnight (NaCl, RNase, PK) and confirmed fragment size between 100-500bp. Chromatin was diluted 7-fold to 0.1%SDS and pre-cleared with 10ul protein G beads with 0.1%BSA for 2hrs 4oC. Then 1/3 of the chromatin was IPed (~5M cells) with 1.5ug Ab, 15uL Dynabeads protein G O/N. Washes were performed twice (5min each, 4oC) with low salt (150mM NaCl), high salt (500mM NaCl), LiCl with 1%SDS & 1% Deoxycholate and TE buffer. After elution and reverse crosslink, input and IP fractions were subjected to qPCR.
Included are an example of the CHIP-qPCR with all primers extracted from previous publications doing the same protein. Western blot using the same Ab confirmed >90% in KO. I have repeated CHIP on other KO cells of a different gene but showing similar problems. However if I run histone markers, the binding in WT cells appears to match previous publications.
Any help is appreciated.
Thankyou in advance.
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