Computational protocol: Expansion of stochastic expression repertoire by tandem duplication in mouse Protocadherin-α cluster

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Protocol publication

[…] The genomic DNA was prepared with the QIAamp DNA Micro Kit (Qiagen) from the cerebellum of a 4-week-old or 3-month-old mouse, or from sperm, or with the EpiTect Plus LyseAll Kit (Qiagen) from the brain of an E12.5 embryo, head of an E9.5 embryo, E7.5 whole embryo, or blastocyst (E3.5), according to the supplier's recommendations. Sperm was obtained from the cauda epididymides of adult male mice. Embryos were obtained by the natural breeding of Pcdhαwt/dup(2-c2) parents. The morning of the vaginal plug was designated E0.5. Bisulfite conversion was performed with the EpiTect Plus DNA Bisulfite Kit (Qiagen), according to the supplier's recommendations. We used “MethPrimer” (http://www.urogene.org/methprimer/) to design primers for use on bisulfite-treated DNA. The primers used for DNA amplification are listed in . The first PCR program consisted of 95°C for 3 min, 25 cycles of 95°C for 30 s, 60°C for 30 s, 72°C for 30 s, and a final extension of 72°C for 7 min. The second PCR was carried out using the same program as the first, except that 32 cycles were performed. To obtain the methylation profile from the acquired data, we used the web-based tool, “QUMA” (http://quma.cdb.riken.jp/).In the HpaII digestion-mediated DNA methylation analysis, HpaII was used to distinguish between methylated (undigested) and unmethylated (digested) HpaII/MspI sites, whereas MspI digested both methylated and unmethylated HpaII/MspI sites. The HpaII/MspI-digested DNA was subjected to PCR analysis to amplify the SNP-containing regions. We mixed 500 ng of DNA with 10 U HpaII or 10 U MspI, and restriction buffer in a 10-μl reaction volume. Samples were incubated at 37°C overnight, and we used 40 ng of the digested DNA for the PCR reaction. The primers are listed in . The PCR program consisted of 95°C for 3 min, 30 cycles of 95°C for 30 s, 60°C for 30 s, 72°C for 1 min, and a final extension of 72°C for 7 min. The PCR products were sequenced using a standard method. The results shown are representative of two independent experiments. […]

Pipeline specifications

Software tools methPrimer, QUMA
Application BS-seq analysis
Organisms Mus musculus, Homo sapiens