GSM4040397: NIS-2_ChIP-seq; Mus musculus; ChIP-Seq
Cultured D1 cells were collected, fixed in 1% formaldehyde and sonicated to yield DNA fragments of ~300bp. For immunoprecipitation, 40ul of anti-Runx3 Ab were added to 15 mL of diluted, fragmented chromatin; Fragmented chromatin IP with NIS served as control. DNA was purified using QIAquick spin columns (QIAGEN). Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Part# 0801-0303). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3' end. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel.