GSM1670855: Mbd3_KD_Klf4_ChIP; Mus musculus; ChIP-Seq
Cells from RNAi-mediated KD were fixed, washed with ice-cold PBS, and pelleted. Cell pellets were lysed through sonication in a Bioruptor (UCD-200) and supernatants were saved. 30 μL of chromatin was stored overnight at 4C for input samples while the remainder of the chromatin was combined with antibody coupled protein A magnetic beads (NEB) and incubated at 4C overnight with constant rotation. H3 antibody (abcam, ab1791), H2AZ antibody (abcam, ab4174), Klf4 antibody (kind gift from Dr. Ng from the Genome Institute of Singapore) coupled protein A magnetic beads (NEB) were blocked with 5 mg/ml BSA overnight at 4C, prior to incubation with sheared chromatin. Magnetic beads were washed and material was eluted at 65C on a thermomixer. Eluted material was transferred to a new microfuge tube, combined and incubated at 65C overnight to reverse crosslinking. Input DNA was diluted with 170 μl elution buffer and treated similarly. Samples were treated with RNaseA/T1 (Ambion), proteinase K (Ambion), and then PCI extracted. Ethanol precipitated ChIP-encriched DNA was then used for library construction.  Libraries were prepared according to Illumina's instructions. 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 (exo minus) and dATP to yield a protruding 3- 'A' base for ligation of barcoded adapters which have a single 'T' base overhang at the 3’ end. DNA purification on Zymo Research PCR purification columns (Zymo, Irvine, CA) was performed following each enzyme reaction. The adaptor-ligated material was then PCR amplified with KAPA-HiFi polymerase using 16 cycles of PCR before size selection of 200-350 bp fragments on a 2% agarose gel. Libraries with different barcodes were pooled together and single-end sequencing (50 bp) was performed on an Illumina HiSeq2000
GEO Accession
GSM1670855