alias: M_19_0097
title: ChIP Oxidative stress Input  Replicate 2
design: Cells were crosslinked at 1% formaldehyde (Sigma cat. No. F8775) for 10 minutes at room temperature and cross-linking process was quenched using glycine per 125mM final concentration for 5 minutes at room temperature. The cells were then lysed using lysis buffer 1 LB1(50mM HEPES-KOH pH 7.5, 10mM NaCl, 1mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100). The nuclei were then pelleted, collected and washed using lysis buffer 2 LB2 (10mM Tris-HCl pH 8.0, 200 mM NaCl, 1mM EDTA, 0.5 mM EGTA). This was followed by lysis using lysis buffer 3 LB3 (10mM Tris-HCl pH 8.0; 100mM NaCl, 1mM EDTA; 0.5mM EGTA; 0.1% Na-Deoxycholate, 0.5% N-laurylsarcosine). ALL lysis buffer will be supplemented with freshly prepared 1X EDTA free protease inhibitor cocktail(Sigma). Sonication was done for the shearing of the chromatin using BRANSON A250 (four cycles of 1.5 min at 20% of amplitude and 50% of Duty Cycle). The chromatin shearing efficiency was then checked on 2% agarose gel. For each immunoprecipitation reaction, 5 g of antibodies were added to 100g of chromatin DNA equivalents. The protein-antibody immunocomplexes were recovered by the magnetic Dynabeads (protein G, Invitrogen). Washes were carried out for the beads using low salt wash buffer (LS) (0.1% SDS; 2mM EDTA, 1% Triton X-100, 20 mM Tris HCl pH 8.0, 150 mM NaCl), and high salt wash buffer (HS) (0.1% SDS, 2mM EDTA, 1% Triton X-100, 20mM Tris-HCl pH 8.0, 500mM NaCl) respectively. The beads were then resuspended in elution buffer (50mM Tris-HCl pH 8.0, 10mM EDTA, 1% SDS). This was followed by de-crosslinking by the addition of RNase A (0.2 mg/ml) and Proteinase K (0.2 mg/ml) to remove RNA and digest protein respectively.The extracted ChIP-DNA (using ChIP-DNA Clean & Concentrator, Zymo) was ChIP-seq library preparation (MicroPlex Library Preparation Kit v2, Diagenode). Final library was pooled and sequenced at Illumina HiSeq 4000 platform.
identifiers.namespace: SUB8074832
identifiers.id: M_19_0097