GSM1363356: Input, dCTCF RNAi; Drosophila melanogaster; ChIP-Seq
Cells were crosslinked with 1% formaldehyde for 10 min at room temperature.  Crosslinking was quenched with glycine and nuclear lysates were sonicated to generate 200–1000 bp DNA fragments.  Chromatin was pre-cleared overnight at 4oC with protein A sepharose beads and then incubated overnight with antibody at 4oC.  Chromatin was then precipitated with Protein A Sepharose beads for 2 hours at 4oC.  After washing and eluting from beads the crosslinking was reversed and DNA was isolated. To generate sequencing libraries, ChIP DNA was prepared for adaptor ligation by end repair (‘End-It DNA End Repair Kit’  - Epicentre Cat# ER0720) and addition of ‘A’ base to 3’ ends (Klenow 3’-5’ exo- NEB Cat# M0212S). Illumina adaptors (Illumina Cat# PE-102-1001) were titrated according to prepared DNA ChIP sample concentration, and ligated with T4 ligase (NEB Cat# M0202S). Ligated ChIP samples were PCR amplified using Illumina primers  and Phusion DNA polymerase (NEB Cat# F-530L) and size selected for 200-300bp by gel extraction.
GEO Accession
GSM1363356