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|Infected RBCs were crosslinked with 1 % formaldehyde (Catalog number—28908, THERMO Scientific) for 10 min, lysed and sonicated in sonication buffer (10 mM Tris–HCl pH 7.5, 200 mM NaCl, 1 % SDS, 4 % NP-40, 1 mM PMSF) to obtain an average chromatin size of 200–400 bp. Chromatin was pre-cleared using 50 µl of a 50 % protein A Sepharose (GE healthcare) slurry for 1 h at 4 °C with gentle inverting. Immunoprecipitations were carried out in 1 ml of IP buffer (20 mM Tris–HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 1 % Triton-X 100). Three µg antibody was used per 20 µg purified chromatin. Input chromatin was obtained after preclearing by de-crosslinking and purified using the Qiaquick column (Qiagen) according to manufacturer’s instructions. Immunoprecipitations were carried out with inverting at 4 °C for 14–16 h.|
|-Add protease inhibitors to all lysis solutions before use.|
|-Keep cells on ice between the rounds of homogenisations.
- Increase or decrease the homogenization step to maximize the yield of nuclei depending on cell line.
-Do not place ethidium bromide in the agarose gel or the electrophoresis buffer, because of the presence of SDS.
|-It is highly critical that the chromatin is of appropriate size and concentration.|
|-It is important to keep the tissue cold to avoid protein degradation.
-Use fresh formaldehyde that is not past the manufacturer's expiration date.
|-Once in solution, store 1M DTT at -20°C.|