title: GSM5970644: mutant_ATACseq_Rep1; Mus musculus; ATAC-seq
library_construction_protocol: Tissue preparation for ATAC-seq: For ATAC-seq analysis, E11.5 embryos were dissected into ice-cold PBS and associated yolk sacs used for rapid genotyping using the Extract-N-Amp Tissue PCR kit as recommended by the manufacturer (Sigma). During genotyping, the facial prominences were carefully removed from individual embryos using a pair of insulin syringes and placed in a 24-well plate with 1 mL of 1 mg/ml Dispase II (in PBS). The samples were incubated with rocking at 37°C for 30-40 minutes and then the facial ectoderm carefully dissected away from the mesenchyme into ice-cold PBS, as described (H. Li & Williams, 2013). Facial ectoderm was then centrifuged at 4°C, 500 g, for 3 minutes in a 1.5ml Eppendorf tube, washed 1x with ice-cold PBS, and then centrifuged again. Following resuspension in 750 µL of 0.25% trypsin-EDTA, samples were incubated at 37°C for 15 minutes with gentle agitation. Following addition of 750 µL of DMEM with 10% FBS to inhibit further digestion, cells were dissociated by pipetting up and down multiple times with wide orifice pipette tips. Cells were subsequently spun at 300 g for 5 minutes and washed with PBS containing 0.4% BSA, and this step was repeated twice. Finally, the cell pellet was resuspended with 50 µL of PBS and the density of the single cell suspension quantified on a hematocytometer.    ATAC-seq transposition, library preparation, and sequencing: Following genotype analysis of embryos used for facial ectoderm isolation, EDKO (Crect; Tfap2aflox/flox; Tfap2bflox/flox) and control littermate samples lacking Crect (Tfap2aflox/+; Tfap2bflox/+) were used for the ATAC-seq protocol, largely following procedures previously described (Buenrostro et al., 2013; Buenrostro et al., 2015; Corces et al., 2017). Briefly, 50,000 cells from each sample were pelleted at 500 g for 5 minutes at 4°C. The pellet was then resuspended in 50 µL of cold lysis buffer (10 mM Tris-HCl, pH 7.5; 10 mM NaCl; 3 mM MgCl2; 0.1% NP-40, 0.1% Tween-20; 0.01% Digitonin) by gently pipetting ~4 times to release the nuclei which were then incubated on ice for 3 minutes. The sample was next spun at 500 g for 20 minutes at 4°C and the pelleted nuclei resuspended in Tagmentation mix (e.g. 25 µL 2x Nextera TD Buffer, 2.5 µL Nextera TD Enzyme, 0.1% Tween-20, 0.01% Digitonin, up to 50 µL with nuclease-free water) and placed at 37°C for 30 minutes in a thermocycler. Following transposition, samples were purified using the QIAGEN minElute PCR Purification Kit (Qiagen) and eluted with 11 µL of supplied Elution Buffer. Transposed DNA was next indexed with a unique barcoded sequence and amplified prior to sequencing. Briefly, 10 µL of transposed DNA was mixed with the Nextera Ad1 PCR primer as well as a unique Nextera PCR primer (e.g. Ad2.x) and NEBNext HighFidelity 2x PCR Master Mix. Samples were then amplified using the following cycling parameters: [72°C, 5 min], [98°C, 30 sec], [98°C, 10 sec; 63°C, 30 sec; 72°C, 1 min (repeat 10-12 cycles)]. Following cycle 5, an aliquot of sample was removed for Sybr-green based quantification to determine the number of remaining cycles required to reach adequate amounts for sequencing without introducing over-amplification artifacts due to library saturation. Following indexing and amplification, samples were purified using two rounds of AmpureXP bead-based size selection. Library purity, integrity, and size were then confirmed using High Sensitivity D1000 ScreenTape and subsequently sequenced using the Illumina NovaSEQ6000 platform and 150 bp paired-end reads to a depth of ~75x106 reads per sample, carried out by the University of Colorado, Anschutz Medical Campus, Genomics and Microarray Core.