GSM3273082: Evf1_TSTS_H3K4me1; Mus musculus; ChIP-Seq
Native ChIP: Whole ganglionic eminences were dissected from 10 Evf2+/+ and 10 Evf2TS/TS E13.5 embryos. Tissues were pooled for each genotype, triturated by pipetting, and filtered through a cell-strainer capped 5 ml polystyrene round- bottom tube (BD Falcon) to make single-cell suspensions. Cells were split into 1 x 106 cell aliquots, and pelleted through centrifugation at 1000 x g for 10 min. Cell pellets were flash frozen in liquid nitrogen, and stored at -80ºC. Nuclei were isolated using EZ Nuclei Isolation Lysis Buffer (N3408, SIGMA). Chromatin was digested in 2U/μl Micrococcal nuclease (M0247S, NEB) at 37°C for 7 min. The reaction was quenched with EDTA (10 mM final concentration). Triton X-100 and Sodium Deoxycholate were added (0.1% final concentration). Samples were incubated on ice for >15 minutes. Immunoprecipitation buffer (20 mM Tris-HCl pH 8.0, 2 mM EDTA, 150 mM NaCl, 0.1% Triton X-100, 1x Protease inhibitor cocktail, 1 mM PMSF) was added to a final volume of 200 μl and the samples were rotated at 4°C for 1 hour. The chromatin was pre-cleared by rotating at 4°C with 15 μl of Protein G–Agarose beads for 1 hour. After centrifugation to pellet the beads, the supernatant was further pre-cleared by rotating at 4°C with 15 μl rabbit IgG conjugated Protein G–Agarose beads for 1 hour. The pre-cleared chromatin was incubated with rabbit IgG (1 μg), or antibodies targeting histone modifications (1 μg) at 4°C for 1-2 hours with rotation. 15 μl of Protein G-Agarose beads blocked with 1% BSA in 1X PBS were added to each sample and incubated at 4°C overnight with rotation. The beads were pelleted by centrifugation and washed twice with 200 μl Low Salt Wash buffer (20 mM Tris-HCl pH 8.0, 2 mM EDTA, 150 mM NaCl, 1% Triton X-100, 0.1% SDS) and twice with 200 μl High Salt Wash buffer (20 mM Tris-HCl pH 8.0, 2 mM EDTA, 500 mM NaCl, 1% Triton X-100, 0.1% SDS). Immunoprecipitated DNA was eluted in 100 μl of ChIP elution buffer (100 mM NaHCO3, 1% SDS) at 65°C for 1-1.5 hour. The DNA was purified using phenol chloroform extraction and ethanol precipitated. The pellet was resuspended in 10 mM Tris-HCl pH 8.5. The DNA was incubated with 20 mg of RNAse A at 55°C for 1 hour. 40 mg Proteinase K were added and incubated at 55°C for 1 hour. The immunoprecipitated DNA was purified using the Qiaquick PCR Purification Kit. Quantity of ChIP'd DNA was determined using Picogreen Reagent (Quant-iT™ PicoGreen dsDNA Assay Kit, Thermo Fisher P11496) and a fluorometer instrument.  150ng to 1ug of DNA was prepared into Illumina libraries, according to manufacturer's instructions, using the TruSeq Nano DNA Library Prep Kit (Illumina, FC-121-4003). Resulting libraries were deep sequenced, using the Illumina HiSeq2500 system in Rapid Run mode, obtaining between 10M and 15M of 100-bp length, single-end reads per library. Quantity of ChIP'd DNA was determined using Picogreen Reagent (Quant-iT™ PicoGreen dsDNA Assay Kit, Thermo Fisher P11496) and a fluorometer instrument.  150ng to 1ug of DNA was prepared into Illumina libraries, according to manufacturer's instructions, using the TruSeq Nano DNA Library Prep Kit (Illumina, FC-121-4003). Resulting libraries were deep sequenced, using the Illumina HiSeq2500 system in Rapid Run mode, obtaining between 10M and 15M of 100-bp length, single-end reads per library.