GSM8374975: N_SH8h_REF6-HA/pif7-1_set1-IP; Arabidopsis thaliana; ChIP-Seq
Fixation: The sample is fixed with 1% formaldehyde (Sigma, 47608). Fragmentation: Nuclear chromatin is extracted and sonicated to fragmentize it to 100-400 base pairs (bp), 5% is taken out as input, and the rest is used for immunoprecipitation. Immunoprecipitation: The primary antibody is incubated with beads, and then mixed with the fragmented chromatin and incubated at 4°C for 6 hours. Elution: The antibody-magnetic bead-protein-DNA complex is sequentially washed with high salt and low salt buffers, and then the IP (Immunoprecipitated) products are eluted. De-crosslinking: The IP products and input are de-crosslinked with RNase A (NEB, T3018L) and Proteinase K (NEB, P8107S). Purification: The de-crosslinked input DNA and IP DNA are purified using a kit (QIAquick PCR Purification Kit, 28106). Purified Input DNA and IP DNA are used to construct libraries with the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, E7645S) kit.