ATAC-seq of Homo sapiens: Glioblastomastem cells
Cells were harvest and washed in ice-cold PBS followed by centrifugation at 500 g for 5minutes. Cells were resuspended in 50 uL cold lysis buffer (10 mM Tris-HCl pH 7.4, 3 mM MgCl2, 10 mM NaCl, 0.1% Igepal CA-630) and mixed end over end. After centrifugation at 500 x g for 10minutes, cell pellet was resuspended in transposition reaction mix (25L TD-Buffer, 2.5L Tn5, 22.5L water) and incubated at 37C for 30 min with gentle mixing. Immediately following the transposition reaction, MinElute PCR Purification Kit (Qiagen) was used to purify transposed DNA. Finally, the PCR amplification was performed to amplify purified DNA and qPCR-based methods were used to quantify ATAC-seq libraries. The libraries were mixed in equimolar ratios and sequenced on BGISEQ-500 platform. ATAC-Seq raw reads were filtered to remove low-quality or adaptor sequences by SOAPnuke. Cleaned reads were mapped to the reference genome of hg38 with Bowtie2 and generated SAM files were converted to BAM files using Samtools using the view function. BAM files which were converted to BigWig files using Deeptools. MACS2 was used to call peaks for open chromatin regions. IDR was applied to measure the reproducibility of findings identified from replicate samples.