GSM2420056: E12.5_PGC_Ｍale ChIP-Seq; Mus musculus; ChIP-Seq
EGFP-BLIMP1 embryos were obtained with intracytoplasmic sperm injection (ICSI) to oocytes (Kimura, 1995 #3053) and transfer of two-cell-stage embryos into oviducts of the surrogate mother mice. The embryonic male gonads at E12.5 were collected, for which sex was determined with the morphological features, and dissociated into single cells by incubating with 0.05 % trypsin and 0.5 mM EDTA (GIBCO) in PBS at 37 °C for 5 min. The cell suspension was then incubated with mouse anti-SSEA1 antibody conjugated with Alexa Fluor 647 (BioLegend). SSEA1 positive cells were purified with FACS. Chromatin immunoprecipitation was performed as described previously(Kurimoto et al., 2015). Libraries for ChIP_seq were prepared as described (Kurimoto, 2015 Cell Stem Cell) with a modification for application to the Illumina systems. Briefly, ChIP-ed and input DNAs were sheared to an average size of 150 bp by ultra-sonication (Covaris S2). The sheared DNAs were end-repaired, dA-tailed, ligated to an amplification adaptor (P1-T Adaptor/F and Barcode-Internal+12-mer/R), amplified with a 10-cycle PCR using Library PCR primer 1 (Life technologies) and Library PCR primer Barcode001 + Internal adaptor. For sequencing on the Illumina systems, we then added an adaptor and index to the amplified library with two rounds of additional PCRs. The 1st round additional PCR was performed for 6 cycles with Read1-P1 and Read2-internal-adaptor primers. The 2nd round PCR was performed for 4 cycles with Illumina P5-Read1 primer and P7-index N-Read2 primer to produce the index-tagged library DNAs. To exclude the initial constant region consisted of the amplification adaptor sequence (P1-T Adaptor) and maximize the efficient sequencing reads, we designed a custom sequencing primer, Custom-primer-29mer (Supplementary Table S2 of the manuscript), which met sequencing on Illumina platform (Caporaso, 2011 PNAS) in terms of the length, GC content, and melting temperature. The DNAs were then sequenced on the HiSeq 2500 platform (Illumina) in high throughput mode to generate single-end 100-bp reads, as the manufacturer's instruction.
GEO Accession
GSM2420056