TruSeq ChIP Library Preparation Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-Remove End Repair Mix and Resuspension Buffer from -15°C to -25°C storage and thaw them at room temperature.	-5–10 ng ChIP-enriched, fragmented input DNA is recommended. -Carefully collect ChIP DNA samples to make sure that they are free of contaminants.

Upstream tips
-Remove End Repair Mix and Resuspension Buffer from -15°C to -25°C storage and thaw them at room temperature.
Protocol tips
-5–10 ng ChIP-enriched, fragmented input DNA is recommended. -Carefully collect ChIP DNA samples to make sure that they are free of contaminants.

Publication protocol

ChIP technique was done according to Dahl and Collas (2008) protocol. We performed experiment both on mouse pituitary Alpha T3-1 cell line (109 cells) and on ovaries from P7 mice (n = 3). Immunoprecipitation was done using our rabbit polyclonal anti-FOXL2 raised against residues DHDSKTGALHSRLDL (Eurogentec s. a., Belgium). The negative control (mock IP) was prepared by performing the entire immunoprecipitation process with beads only, either including no antibody or an irrelevant antibody. For ChIP-Seq, paired end libraries were prepared according to standard Illumina protocol, size selection was done to obtain 200 nucleotides (nt) long inserts. Sequencing of 21nt long paired ends was achieved with Illumina Genome Analyzer II. ChIP-Seq reads were mapped onto the reference genome mm9 using Bowtie (Langmead et al., 2009). The alignment was performed using the paired-end mode and the number of mismatches was set to 0. MACS1.4 (Zhang et al., 2008) was used to identify the putative FOXL2 binding regions. Samples and negative controls were analysed separately. ChIP regions were characterized with CEAS (Ji et al., 2006) using the RefSeq annotation. Comparing ChIP samples versus mock controls, a significative enrichment of ChIP regions was found to be related to promoter regions. ChIP regions located from 0 to 1000 nt upstream of the TSS were used to perform a motif analysis using MEME-ChIP (Machanick and Bailey, 2011). For both the pituitary Alpha T3-1 cell line and P7 ovaries, the same motif was found to be enriched. Using an in-house developed software, we identified the ChIP regions containing the motif obtained by MEME-ChIP in order to predict the putative FOXL2 binding sites in the long-range Col1a2 promoter region.

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