GSM2058753: LNCaP_siLUC_2MK4_replicate 2; Homo sapiens; ChIP-Seq
For each ChIP reaction, 3 million formaldehyde cross-linked cells were lysed and sonicated with a digital probe sonicator to shear chromatin. 5µg of anti-dimethylated histone H4 Lysine 4 were used to immunoprecipitate DNA overnight at 4°C. One percent of chromatin was used as input. Immune complexes were collected with 30 µl protein A magnetic beads. After extensive washing, immune complexes were eluted, cross-links were reversed, and DNA from 4 replicate samples were pooled and  purified with miniElute PCR purification kit and eluted with 20 µl of elution buffer. 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. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols.
GEO Accession
GSM2058753