GSM2102240: Mbd2KD_Mbd3ChIP_rep1; 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 4°C for input samples while the remainder of the chromatin was combined with antibody coupled protein A magnetic beads (NEB) and incubated at 4°C overnight with constant rotation. Mbd3 antibody (Bethyl) coupled protein A magnetic beads (NEB) were blocked with 5 mg/ml BSA overnight at 4°C, prior to incubation with sheared chromatin. Magnetic beads were washed and material was eluted at 65°C on a thermomixer. Eluted material was transferred to a new microfuge tube, combined and incubated at 65°C 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-) 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 1% agarose gel. Libraries with different barcodes were pooled together and single-end sequencing was performed on an Illumina HiSeq2000.
GEO
GSM2102240