ATAC-seq Protocol for CD4+ T Cells

The ATACseq libraries were prepared based on the Omni-ATACseq protocols with some minor modifications(82 (link)). Briefly, 60,000 FACS-sorted CD4⁺ T cells were used to prepare nuclei. The transposition reaction was performed by incubating nuclei with Nextera Tn5 transposase (Illumina) at 37 °C for 1hr. The transposition mixture was purified using a Zymo DNA Clean and Concentrator kit. The ATAC libraries were amplified for 11–13 cycles using NEBNext 2X MasterMix and Nextera Index primers. The amplified libraries were size-selected using AMPure beads to remove the large fragment of DNA and then purified using Zymo DNA Clean and Concentrator kit. Six ATAC libraries were pooled and sequenced on a NextSeq500 instrument in a pair-end 75 cycle run. 60–100 million read pairs were obtained for each sample.

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Ding Z.C., Shi H., Aboelella N.S., Fesenkova K., Park E.J., Liu Z., Pei L., Li J., McIndoe R.A., Xu H., Piazza G.A., Blazar B.R., Munn D.H, & Zhou G. (2020). Persistent STAT5 activation reprograms the epigenetic landscape in CD4+ T cells to drive polyfunctionality and antitumor immunity. Science immunology, 5(52), eaba5962.

Publication 2020

Nextera Tn5 transposase Zymo DNA Clean and Concentrator kit

Atac Cd4 t cells Nuclei Primers Tn5 transposase

Corresponding Organization : Augusta University

Other organizations : Nanchang University, USA Mitchell Cancer Institute, University of South Alabama, University of Minnesota

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Variable analysis

independent variables

The transposition reaction, where nuclei were incubated with Nextera Tn5 transposase (Illumina) at 37 °C for 1hr

dependent variables

ATAC libraries were sequenced on a NextSeq500 instrument in a pair-end 75 cycle run, with 60–100 million read pairs obtained for each sample

control variables

60,000 FACS-sorted CD4+ T cells were used to prepare nuclei
The transposition mixture was purified using a Zymo DNA Clean and Concentrator kit
The ATAC libraries were amplified for 11–13 cycles using NEBNext 2X MasterMix and Nextera Index primers
The amplified libraries were size-selected using AMPure beads to remove the large fragment of DNA and then purified using Zymo DNA Clean and Concentrator kit

controls

No explicit positive or negative controls were mentioned in the protocol.

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