title: GSM3521706: kdrl:mCherry+mrc1a:GFP-_Rep 3; Danio rerio; ATAC-seq
library_construction_protocol: 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
attributes.tag: GEO Accession
attributes.value: GSM3521706