title: GSM5006044: 4Cseq Dlx5/6UCE bait Sox2flfl cre neg; Mus musculus; OTHER
library_construction_protocol: 4C was performed as previously described (van de Werken et al. 2012), with some modifications. GEs from Sox2 fl/fl Cre+ and Sox2 fl/fl Cre- single embryos were dissected in L15 and kept in separate tubes. A single cell suspension was obtained through gentle pipetting of the tissue in 250 μl of L15. Cells (approximately 2 x106 cells per embryo) were transferred to a tube containing 5 ml 2% paraformaldehyde/10% Fetal Bovine Serum (FBS) and incubated with rotation for 10 min at room temperature. 710 μl of 1M glycine were added to quench the formaldehyde and tubes were placed on ice. Cells were pelleted by centrifugation at 1300 rpm for 8 min at 4°C. The supernatant was removed and cells were gently resuspended in 2.5 ml ice-cold 4°C Lysis Buffer (50 mM Tris pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, 1% Triton X-100, protease inhibitors) and incubated for 10 min on ice.  Cells were pelleted by centrifugation at 1800 rpm for 5 min at 4°C. The supernatant was removed, and the cells were washed with 1 ml ice-cold 1x PBS by gentle resuspension. Cells were transferred to 1.5 ml microcentrifuge tubes and centrifuged at 2,400 rpm for 2 min at 4°C. The supernatant was removed and the cell pellets were flash frozen in liquid nitrogen and stored at -80°C.   The cell pellets were resuspended in 440 μl of molecular grade water and 60 μl of Cutsmart Buffer (New England Biolabs) were added. Tubes were incubated at 37°C and 15 μl of 10% SDS were added. Samples were incubated for 1 hour at 37°C while shaking at 900 rpm. 75 ul of 20% Triton X-100 were added to the samples and incubated at 1 hour at 37°C while shaking at 900 rpm. 200 U of EcoRI-HF were added to the samples and incubated overnight at 37°C while shaking. The next day, 200 U of EcoRI-HF were added and incubated overnight at 37°C with shaking. Complete digestion was confirmed by agarose gel electrophoresis. If undigested DNA was still present, another 200 U of restriction enzyme was added and incubated overnight. The enzyme was inactivated at 65°C for 20 min. Samples were transferred to a 15 ml conical tube. 100 U of T4 DNA Ligase were added (2 ml reaction volume) and incubated overnight at 16°C. The next day, 500 μl of molecular grade water, 50 μl fresh T4 ligase buffer and 100 U of T4 DNA ligase were added to the samples and incubated overnight at 16°C. Complete ligation was confirmed by agarose gel electrophoresis. The DNA was extracted using standard ethanol precipitation procedures (van de Werken et al. 2012).    The DNA pellet was resuspended in 450 μl of molecular grade water, then 50 μl of DpnII buffer and 50 U of DpnII were added and incubated at 37°C overnight. Complete digestion was confirmed by agarose gel electrophoresis. The enzyme was inactivated at 65°C for 20 min. Samples were transferred to a 50 ml conical tube and ligation was performed using 100 U of T4 DNA ligase in a total volume of 3.5 ml at 16°C overnight. The DNA was precipitated using standard ethanol precipitation procedures. The samples were purified using the Qiaquick PCR purification kit (2 columns per sample). 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, as follows, 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). mouse E13.5 ganglionic eminences were dissected, and single cell suspensions were processed for CUT&RUN, mouse E13.5 ganglionic eminences were dissected, and  processed for 4Cseq