ATAC-seq Protocol for Chromatin Profiling

ATAC-seq was performed as previously described (Buenrostro et al. 2013 (link)). Sixty-thousand cells were washed once with 100 mL of PBS and resuspended in 50 mL of lysis buffer (10 mM Tris-HCl at pH 7.4, 10 mM NaCl, 3 mM MgCl₂, 0.2% IGEPAL CA-630). The suspension of nuclei was then centrifuged at 500g for 10 min at 4°C, followed by the addition of 50 mL of transposition reaction mix (25 mL of TD buffer, 2.5 mL of Tn5 transposase, 22.5 mL of nuclease-free H₂O) and incubation for 30 min at 37°C. DNA was isolated using MiniElute kit (Qiagen). Libraries were amplified by PCR (13 cycles). After the PCR reaction, the library was selected for fragments between 100 and 1000 bp with AMPure XP beads (Beckman Coulter). Libraries were purified with Qiaquick PCR (Qiagen) and integrity-checked on a Bioanalyzer before sequencing.

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Yagi M., Ji F., Charlton J., Cristea S., Messemer K., Horwitz N., Di Stefano B., Tsopoulidis N., Hoetker M.S., Huebner A.J., Bar-Nur O., Almada A.E., Yamamoto M., Patelunas A., Goldhamer D.J., Wagers A.J., Michor F., Meissner A., Sadreyev R.I, & Hochedlinger K. (2021). Dissecting dual roles of MyoD during lineage conversion to mature myocytes and myogenic stem cells. Genes & Development, 35(17-18), 1209-1228.

Publication 2021

MiniElute kit AMPure XP beads Qiaquick PCR

Atac seq Buffer Cells Igepal ca 630 Library Mgcl2 Nacl Nuclei Tn5 transposase Tris

Corresponding Organization :

Other organizations : Harvard University, Massachusetts General Hospital, Harvard Stem Cell Institute, Broad Institute, Massachusetts Institute of Technology, Max Planck Institute for Molecular Genetics, Brigham and Women's Hospital, Dana-Farber Cancer Institute, Joslin Diabetes Center, University of Connecticut

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Variable analysis

independent variables

Number of cells used (60,000 cells)

dependent variables

DNA fragments between 100 and 1000 bp obtained after library preparation

control variables

Lysis buffer composition (10 mM Tris-HCl at pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.2% IGEPAL CA-630)
Centrifugation conditions (500g for 10 min at 4°C)
Transposition reaction mix composition (25 mL of TD buffer, 2.5 mL of Tn5 transposase, 22.5 mL of nuclease-free H2O)
Transposition reaction incubation time and temperature (30 min at 37°C)
DNA isolation using MiniElute kit (Qiagen)
Library amplification by PCR (13 cycles)
Library fragment selection (100-1000 bp) using AMPure XP beads
Library purification using Qiaquick PCR (Qiagen)
Library integrity check on a Bioanalyzer

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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