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Chromatin Preparation

Step 1: Crosslinking
(Stabilization of Protein-DNA complexes) with 1% formaldehyde, and glycine solution for quenching

Step 2: Cell Lysis
with Hypotonic Buffer

Step 3: DNA Fragmentation by Enzymatic Digestion
with Digestion buffer, lysis buffer, PIC, shearing cocktail, enzymatic stop solution, DNA sample quality and quantity checking

Immunopreciptation

Step 4: Chromatin Immunoprecipitation
with Go-ChIP-Grade™ Purified Antibody, Isotype Control Antibody

Step 5: Column Conditioning, Washing and Elution
with wash buffers and elution buffer

Step 6: Reverse Crosslinking
with 1M NaHCO3, 5M NaCl

Downstream DNA Analysis

Step 7: Digestion with Proteinase K and DNA Purification
with Proteinase K, Proteinase K stop solution, DNA purification cols and buffers

Step 8: qPCR to quantify DNA
with primers to the target gene of interest

Step 9: Data Acquisition and Analysis
(The amount of IP'ed DNA in each sample is represented as signal relative to the 5% of total amount of input chromatin)