GSM3273109: Evf2_WT.4C; Mus musculus; OTHER
Whole ganglionic eminences (GEs) 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 fixed in 2% paraformaldehyde/10% Fetal Bovine Serum (FBS) at room temperature for 10 min with rotation. 125 mM glycine was used to quench the formaldehyde. The 4C method used has been described in detail (van de Werken et al. 2012) EcoRI was used for the primary restriction digestion and DpnII was used for the secondary restriction digestion. The following steps were performed to generate the 4C library for sequencing. First, overhangs were added to the 4C template using PCR amplification with primers containing the bait sequence. Primers: Dlx5/6UCE Forward: 5'TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGATGCCAAACCACTGTGAGTGTA3', Dlx5/6UCE Reverse: 5'GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGTCCCCAATGTCTGCTTCAA 3'. PCR reaction: 200 ng 4C template, 0.2 mM dNTPs, 35 pmol Primer Dlx5/6UCE-Fwd, 35 pmol Primer Dlx5/6UCE-Rev, 1.75 U Expand Long Template Enzyme Mix (Roche), 1X Buffer I. PCR cycles: 94°C - 2 min, 94°C – 10 sec, 55°C - 1min, 68°C - 3 min, 29 cycles, 68°C - 5min. The PCR product was purified using the High Pure PCR Product Purification Kit (Roche). Then, the 4C DNA containing the overhangs was used as template for a second PCR that adds index sequences and Illumina sequencing adapters to generate the 4C library for sequencing. PCR reaction (50 μl): 225 ng DNA template, 0.5 mM dNTPs, 5μl Nextera XT Index1 primer (N7XX, Illumina), 5 μl Nextera Index 2 primer (S5XX, Illumina), 3.5 U Expand Long Template Enzyme Mix (Roche), 1X Buffer I. PCR cycles: 94°C - 5 min, 94°C – 10 sec, 55°C – 30 sec, 68°C - 1 min, 8 cycles, 68°C - 7min.The PCR product was purified using the High Pure PCR Product Purification Kit (Roche). 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.