title: GSM3083211: SKMel-239_SIRT6.2-7_ATACseq; Homo sapiens; ATAC-seq
library_construction_protocol: All ATAC-seq libraries were prepared essentially as previously described with modifications43. Briefly, 50k cells were resuspended in 1mL of cold ATAC-seq resuspension buffer (RSB; 10 mM Tris-HCl pH 7.4, 10 mM NaCl, and 3 mM MgCl2 in water). After centrifugation, cell pellets were resuspended in 50 µL of ATAC-seq RSB containing 0.1% NP40, 0.1% Tween-20, and 0.01% digitonin. This cell lysis reaction was incubated on ice for 3 min. Nuclei were isolated using 1 mL of ATAC-seq RSB containing 0.1% Tween-20 (without NP40 or digitonin) and centrifugation. Nuclei were resuspended in 50 μl of transposition mix (25 μl 2× TD buffer43, 2.5 μl transposase44 (100 nM final), 16.5 μl PBS, 0.5 μl 1% digitonin, 0.5 μl 10% Tween-20, and 5 μl water). Transposition reactions were incubated at 37 °C for 30 min in a thermomixer with shaking. Reactions were cleaned up with Qiagen miniElute PCR Purification columns. ATAC-seq libraries were PCR amplified with 5 to 7 cycles, size selected (100-800bp), and purified using XPure magnetic beads45. Libraries were sequenced on NextSeq500 (75bp paired-end reads). Reads were aligned to the GRCh37/hg19 using Bowtie2 (version 2.1.0) with parameters –S –X 200046. Reads aligned to mtDNA, with quality value Q < 30, as well as duplicated reads, were removed using in-house scripts and Samtools. Significant peaks were identified using MACS2 with parameters –nomodel –nolambda –keepdup all –slocal 10000. Coverage tracks were generated using deepTools bamCoverage with parameters –normalizeUsingRPKM –binsize 10. All ATAC-seq libraries were prepared essentially as previously described with modifications43. Briefly, 50k cells were resuspended in 1mL of cold ATAC-seq resuspension buffer (RSB; 10 mM Tris-HCl pH 7.4, 10 mM NaCl, and 3 mM MgCl2 in water). After centrifugation, cell pellets were resuspended in 50 µL of ATAC-seq RSB containing 0.1% NP40, 0.1% Tween-20, and 0.01% digitonin. This cell lysis reaction was incubated on ice for 3 min. Nuclei were isolated using 1 mL of ATAC-seq RSB containing 0.1% Tween-20 (without NP40 or digitonin) and centrifugation. Nuclei were resuspended in 50 μl of transposition mix (25 μl 2× TD buffer43, 2.5 μl transposase44 (100 nM final), 16.5 μl PBS, 0.5 μl 1% digitonin, 0.5 μl 10% Tween-20, and 5 μl water). Transposition reactions were incubated at 37 °C for 30 min in a thermomixer with shaking. Reactions were cleaned up with Qiagen miniElute PCR Purification columns. ATAC-seq libraries were PCR amplified with 5 to 7 cycles, size selected (100-800bp), and purified using XPure magnetic beads45. Libraries were sequenced on NextSeq500 (75bp paired-end reads). Reads were aligned to the GRCh37/hg19 using Bowtie2 (version 2.1.0) with parameters –S –X 200046. Reads aligned to mtDNA, with quality value Q < 30, as well as duplicated reads, were removed using in-house scripts and Samtools. Significant peaks were identified using MACS2 with parameters –nomodel –nolambda –keepdup all –slocal 10000. Coverage tracks were generated using deepTools bamCoverage with parameters –normalizeUsingRPKM –binsize 10.
attributes.tag: GEO Accession
attributes.value: GSM3083211