GSM3521701: kdrl:mCherry+mrc1a:GFP+_Rep 1; Danio rerio; ATAC-seq
FACS-isolated cells were spun at 500 x g for 5 min at 4° C. Cells were washed once with 50 uL of cold 1X PBS and spun down at 500 x g for 5 min at 4° C. After discarding supernatant, cells were lysed using 50 uL cold lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA-360). Nuclei were spun down at 500 x g for 10 mins at 4° C. Nuclei were kept on ice and subsequently resuspended in 25 uL 2X TD Buffer (Illumina Nextera kit), 2.5 uL Transposase enzyme (Illumina Nextera kit, 15028252) and 22.5 uL Nuclease-free water in a total of 50 uL reaction. Transposase reaction was continued for 1 hr at 37° C. Immediately following the reaction, DNA was purified using Qiagen MinElute PCR purification kit (28004) in a final volume of 10 uL. Libraries were purified according to Illumina protocol using the transposed DNA, NEB PCR master mix, Sybr green, universal and library-specific Nextera index primers. First round of PCR cycles was performed with the following cycles: 72° C, 5 min; 98° C, 30 sec; [98°C, 10 sec; 63 °C, 30 sec; 72 °C, 1 min] X 5 cycles; hold at 4°C. Reactions were kept on ice and using a 5 uL reaction aliquot, appropriate number of additional cycles required for further amplification were determined in a side qPCR reaction with the following procedure: 98 °C , 30 sec; [98 °C, 10 sec; 63 °C, 30 sec; 72 °C, 1 min] X 20 cycles; hold at 4 °C. Upon determining the additional number of PCR cycles required further for each sample, library amplification was conducted using the following conditions: 98°C, 30 sec; [98°C,10 sec; 63°C, 30 sec; 72 °C, 1 min] X appropriate number of cycles; hold at 4°C. Libraries prepared went through quality control analysis using an Agilent Bioanalyzer. Samples with appropriate nucleosomal laddering profiles were selected for next generation sequencing using Illumina Hiseq 2500 platform
GEO Accession
GSM3521701