ATAC-seq of 20,000 sorted cells

ATAC-seq was carried out as described earlier with minor modification (31 (link)). 20 000 cells were sorted in ice-cold PBS. Nuclei were isolated with ATAC-Lysis Buffer (10mM Tris-HCl pH7.4, 10mM NaCl, 3mM MgCl₂, 0.1% IGEPAL) and were used for tagmentation using Nextera DNA Library Preparation Kit (Illumina) from 2–3 biological replicates. After tagmentation DNA was purified with MinElute PCR Purification Kit (Qiagen). Tagmented DNA was amplified with Kapa Hifi Hot Start Kit (Kapa Biosystems) using 9 PCR cycles. Amplified libraries were purified again with MinElute PCR Purification Kit. Fragment distribution of libraries was assessed with Agilent Bioanalyzer and libraries were sequenced on a HiSeq 2500 platform.

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Patsalos A., Tzerpos P., Halasz L., Nagy G., Pap A., Giannakis N., Lyroni K., Koliaraki V., Pintye E., Dezso B., Kollias G., Spilianakis C.G, & Nagy L. (2019). The BACH1-HMOX1 regulatory axis is indispensable for proper macrophage subtype specification and skeletal muscle regeneration. Journal of immunology (Baltimore, Md. : 1950), 203(6), 1532-1547.

Publication 2019

Nextera DNA Library Preparation Kit MinElute PCR Purification Kit Kapa Hifi Hot Start Kit

Atac Atac seq Biological Buffer Cells Cold Dna library Mgcl2 Nacl Nuclei Tris

Corresponding Organization : Johns Hopkins All Children's Hospital

Other organizations : University of Crete, Alexander Fleming Biomedical Sciences Research Center, Foundation for Research and Technology Hellas, National and Kapodistrian University of Athens, Jósa András Múzeum

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Variable analysis

independent variables

Cell sorting (20,000 cells sorted in ice-cold PBS)

dependent variables

ATAC-seq data

control variables

ATAC-Lysis Buffer (10mM Tris-HCl pH7.4, 10mM NaCl, 3mM MgCl2, 0.1% IGEPAL) used for nuclei isolation
Nextera DNA Library Preparation Kit (Illumina) used for tagmentation
MinElute PCR Purification Kit (Qiagen) used for DNA purification
Kapa Hifi Hot Start Kit (Kapa Biosystems) used for library amplification with 9 PCR cycles
Agilent Bioanalyzer used for library fragment distribution assessment
HiSeq 2500 platform used for library sequencing

controls

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

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