GSM3273103: Evf2_WT_Input_10; Mus musculus; ChIP-Seq
Crosslinked 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. For anti-DLX and anti-H3K27ac ChIP cells were fixed in 1% paraformaldehyde for 10 min. For anti-SMC1 and anti-SMC3 ChIP cells were fixed in 1% paraformaldehyde for 90 min. Cells were lysed in SDS lysis buffer (1% SDS, 50 mM Tris-HCl pH 8, 10 mM EDTA) with protease inhibitors (11836153001, Roche). The lysates were sonicated with a Bioruptor Pico (Diagenode) for 10 cycles (30 sec On, 30 sec Off). The lysates were then centrifuged to pellet cellular debris and the supernatant collected for ChIP. 25 μg of chromatin were diluted 1:10 in RIPA Buffer (10mM Tris pH 7.6, 1mM EDTA, 0.1% SDS, 0.1% Sodium Deoxycholate, 1% Triton X-100) with protease inhibitors (B14002, Biotool). The chromatin was pre-cleared by rotating at 4°C with 50 μl of Protein G–Agarose beads (11719416001, Roche) for 1 hour. After centrifugation to pellet the beads, the supernatant was further pre-cleared by rotating at 4°C with 50 μl rabbit IgG conjugated Protein G–Agarose beads for 1 hour. The pre-cleared chromatin was incubated with rabbit IgG (2.5 μg), previously validated anti-pan-DLX (2.5 μg, (Feng et al. 2006; Bond et al. 2009; Cajigas et al. 2015)), anti-H3K27ac (1 μg, Abcam Ab4729), anti-SMC1 (1 μg, Bethyl A300-055A) or anti-SMC3 (1 μg, Abcam ab9263) at 4°C for 4 hours with rotation. 50 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. Beads were pelleted by centrifugation and washed twice with Low Salt Buffer (20 mM Tris-HCl pH 8.1, 2 mM EDTA, 150 mM NaCl, 0.1% SDS, 1% Triton X-100), three times with High Salt Buffer (20 mM Tris-HCl pH 8.1, 2 mM EDTA, 500 mM NaCl, 0.1% SDS, 1% Triton X-100), four times with LiCl buffer (0.25M LiCl, 10 mM Tris-HCl pH 8.1, 1 mM EDTA, 1% sodium deoxycholate and 1% NP-40), twice with 0.1% Tween-20 in 1X PBS, and once with TE buffer (10 mM Tris-HCl pH 8.1 and 1 mM EDTA). Immunoprecipitated DNA was eluted from the beads by incubation with 200 μl of elution buffer (50 mM Tris-HCl pH 8, 10 mM EDTA, 1% SDS) at 65°C for 1 hour. The beads were removed by centrifugation and DNA crosslinking was reversed at 65°C for 4 hours. The DNA was incubated with 20 mg of RNAse A at 55°C for 1 hour. 40 mg Proteinase K (3115879001, Roche) were added and incubated at 55°C for 1 hour. The Immunoprecipitated DNA was purified using the Qiaquick PCR Purification Kit (Qiagen). 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.