title: ATAC-seq of Embryonic Emu Forelimb Bud (HH25)
design: Single cell suspensions were generated as follows: tissue was dissected from each embryo in cold PBS.  Tissue was immediately transferred to 1x Trypsin in EDTA Solution (Sigma) for 10-15 minutes at room temperature. Following trypsinization, the tissue was transferred to a neutralizing culture media (DMEM (Gibco) with 10% FBS (Gibco) and 1% Pen Strep (Gibco)) and pipette-mixed until homogenized. Homogenate was filtered through 35m nylon mesh filters and filtrate was taken immediately to cell counting. Cells were counted on an inverted light microscope using a hemocytometer. Cells to be counted were diluted in Trypan Blue (Sigma) to distinguish living cells from dead. Approximately 25,000-50,000 live undiluted cells were isolated and utilized for each library. Nucleus isolation, transposition, and reaction cleanup were carried out with no modifications. Transposition utilized the Tn5 transposase and buffer from the Nextera DNA Library Preparation Kit (Illumina). Following transposition reaction cleanup, all libraries were PCR amplified for 11 cycles.  PCR reactions were cleaned using the MinElute PCR Purification Kit (Qiagen) or via bead purification as follows.  45 ul of product was combined with 18 ul (or 0.4x) of AmpureXP beads (Beckman Coulter) to remove large fragments, followed by 63 ul or 1.4x to remove small fragments). Libraries were visualized on a HS DNA Bioanalyzer Chip (Agilent) or hs5000 TapeStation (Agilent) and quantified using a Library Quantification Kit (KAPA Biosystems) for multiplexing. ATAC-seq libraries were sequenced as 75 bp paired-end reads on NextSeq High 150 flowcells (Illumina).