Computational protocol: Inhibition of CD200R1 expression by C/EBP beta in reactive microglial cells

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Protocol publication

[…] qChIP was performed as described by Straccia et al. [] with some modifications. Briefly, control and LPS-treated (4 hours) primary mixed glial cultures or BV2 cells were cross-linked with formaldehyde (1% vol/vol final concentration) and processed for chromatin extraction. Chromatin shearing resulted in fragments of 500 bp. An aliquot of chromatin sheared from each sample was separated as a loading control for the experiment (input). The rest of the sample was processed for chromatin immunoprecipitation (ChIP) using Dynabeads® protein A (Invitrogen) and 2 μg of polyclonal rabbit anti-C/EBPβ antibody (Santa Cruz Biotechnology) or rabbit IgG (Santa Cruz Biotechnology) as the negative control. DNA was isolated after the elimination of the protein from the immunoprecipitated samples. Input and ChIP samples were analyzed using real-time PCR and SYBR green (Bio-Rad). Three microliters of input DNA (diluted 1/50) and ChIP samples were amplified in triplicate in 96-well plates using the MyIQ Bio-Rad Real Time Detection System, as described in the section on quantitative real-time PCR. The C/EBPβ binding site in the IL-10 promoter was used as a positive control (Liu et al., 2003). Match-1.0 (public version, BioBase) and MatInspector (Genomatix) were used to identify C/EBPβ consensus sequences in the 5,000 bp region upstream from the ATG translation start site of the CD200R1 gene. The sequences for each amplified locus and the primers used are shown in Table . Samples were run for 45 cycles (95°C for 30 seconds, 62°C for one minute, and 72°C for 30 seconds). For details regarding data analysis see the section on quantitative real-time PCR. […]

Pipeline specifications

Software tools Biobase, MatInspector
Application Miscellaneous
Organisms Mus musculus