Name: mapping genome-wide accessible chromatin in primary human t lymphocytes by atac-seq
Uploaded: 2017-11-13T13:00:00
Description: Watch this Scientific Journal Video about Mapping Genome-wide Accessible Chromatin in Primary Human T Lymphocytes by ATAC-Seq at JoVE.com

This work is supported by the Israel Science Foundation (grant 748/14), Marie Curie Integration grant (CIG)- FP7-PEOPLE-20013-CIG-618763 and I-CORE Program of the Planning and Budgeting Committee and The Israel Science Foundation grant no. 41/11.

All the procedures were approved by institutional review board of Bar Ilan University and the protocol follows guidelines provided by the committee approving the experiments.
1. Purification of Na&#239;ve Human CD4+ Cells and Polarization to T Helper 1 (Th1) and Th2 Cells
Note: Here we describe the procedure starting from frozen human peripheral blood mononuclear cells (PBMCs). The first step consists of isolating CD4+ cells using microbeads and columns that usually g...

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The ATAC-seq protocol described here has been successfully employed for the analysis of accessible chromatin in primary cells (human Th1, Th2 cells, and B cells) as well as the cultured cell lines (MCF10A human breast cancer cells and U261 glioblastoma cells). Applying ATAC-seq to other cell types may require some protocol optimization, especially in the lysis step. If the concentration of non-ionic detergent is too high, there may be a higher percentage of mitochondrial DNA contamination. This can be reduced by decreasi...

ATAC-seq1,2 is a robust method that enables identification of regulatory3 open chromatin regions and nucleosome positioning. This information is applied for inferring the location, identity, and activity of transcription factors. The method&#39;s sensitivity for measuring quantitative variations in chromatin structure allows the study of the activity of chromatin factors, including chromatin remodelers and modifiers, as well as the transcriptional activity of RNA polymerase II1. Thus ATAC-seq provides a powerful and unbiased approach for deciphering mechanisms that govern transcriptional regulation in any cell type of interest. We describe the adaptation of ATAC-seq to primary human Th1 and Th2 cells.
In ATAC-seq, hyperactive Tn5 transposase loaded with adaptors for next-generation sequencing (NGS) couples DNA fragmentation with tagging of DNA with adaptors (i.e., the &#34;tagmentation&#34; process)1. Following PCR amplification, the resulting DNA libraries are ready for next-generation sequencing (Figure 1). The preferential tagmentation of accessible chromatin is detected by the analysis of local enrichment of ATAC-seq sequencing reads.
The short experimental procedure and requirement for less starting material, relative to other methods for measuring chromatin accessibility and nucleosomal positioning such as DNase-seq4, FAIRE-seq5, and MNase-seq6, has promoted the use of ATAC-seq in multiple biological systems including human primary cells1,7 and clinical samples8, as well as unicellular organisms9, plants10, fruit flies11, and various mammals12.
The identity of transcription factors which are bound to accessible loci can be uncovered by analyzing the enrichment of their binding sequence motifs or combining ATAC-seq with chromatin immunoprecipitation (ChIP) followed by high-throughput DNA sequencing (ChIP-seq). This approach enabled the identification of lineage-specific transcription factors important for hematopoiesis in mouse13. The unbiased and global nature of ATAC-seq allows studying gene regulation in organisms for which reagents such as antibodies for ChIP analysis are not available. For example, evolutionary variations in cis-regulatory regions have been identified by studying cranial neural crest cells from humans and chimpanzees14, developmental variations in regulatory elements during early mouse embryogenesis15, changes in the regulatory landscape during a life cycle of unicellular C. owzarzaki9, and the evolution of promoters and enhancers across 20 mammal species12.
ATAC-seq has also been instrumental for measuring chromatin accessibility in single cells, thus revealing variability within cell populations, which usually evades genome-wide studies7,16. In addition, ATAC-seq can be used to study changes that occur in DNA regulatory regions in disease conditions, in which samples are rare. For example, ATAC-seq can be used to study changes in the regulatory landscape during the onset of acute myeloid leukemia (AML)17 or Ras-driven oncogenesis11.

mapping genome-wide accessible chromatin in primary human t lymphocytes by atac-seq

Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) is a method used for the identification of open (accessible) regions of chromatin. These regions represent regulatory DNA elements (e.g., promoters, enhancers, locus control regions, insulators) to which transcription factors bind. Mapping the accessible chromatin landscape is a powerful approach for uncovering active regulatory elements across the genome. This information serves as an unbiased approach for discovering the network of relevant transcription factors and mechanisms of chromatin structure that govern gene expression programs. ATAC-seq is a robust and sensitive alternative to DNase I hypersensitivity analysis coupled with next-generation sequencing (DNase-seq) and formaldehyde-assisted isolation of regulatory elements (FAIRE-seq) for genome-wide analysis of chromatin accessibility and to the sequencing of micrococcal nuclease-sensitive sites (MNase-seq) to determine nucleosome positioning. We present a detailed ATAC-seq protocol optimized for human primary immune cells i.e. CD4+ lymphocytes (T helper 1 (Th1) and Th2 cells). This comprehensive protocol begins with cell harvest, then describes the molecular procedure of chromatin tagmentation, sample preparation for next-generation sequencing, and also includes methods and considerations for the computational analyses used to interpret the results. Moreover, to save time and money, we introduced quality control measures to assess the ATAC-seq library prior to sequencing. Importantly, the principles presented in this protocol allow its adaptation to other human immune and non-immune primary cells and cell lines. These guidelines will also be useful for laboratories which are not proficient with next-generation sequencing methods.

The final outcome of this protocol is an ATAC-seq library of typically 3 - 20 ng/&#181;L. When run on a system for DNA integrity analysis (see Table of Materials/Equipment), they show ladder-like appearance2 (Figure 3A). The average size of DNA fragments is typically ~450 - 530 bp.
Proper quality control of the ATAC-seq libraries prior to performing next-gene...

Watch this Scientific Journal Video about Mapping Genome-wide Accessible Chromatin in Primary Human T Lymphocytes by ATAC-Seq at JoVE.com

Mapping Genome-wide Accessible Chromatin in Primary Human T Lymphocytes by ATAC-Seq

Assay for Transposase-Accessible Chromatin coupled with high-throughput sequencing (ATAC-seq) is a genome-wide method to uncover accessible chromatin. This is a step-by-step ATAC-seq protocol, from molecular to the final computational analysis, optimized for human lymphocytes (Th1/Th2). This protocol can be adopted by researchers without prior experience in next-generation sequencing methods.

Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) is a method used for the identification of open (accessible) regions ...

The overall goal of this high-throughput method using transposase is to identify accessible chromatin regions in the human genome. This method can help answer key questions in the epigenomic fields such as genome-wide locations of regulatory elements. The main advantages of this technique are that it is robust, relatively short and requires less starting material relatively to other methods of measuring chromatin accessibility such as DNase-seq or FAIRE-seq.Though this method can provide insight into regulatory landscape of human T lymphocytes, it can also be applied to other systems such as other human primary cells, cancer cells, human clinical samples, cells from other mammals and organisms. Thaw a one-milliliter aliquot of 10 million human PBMCs and transfer it to a 50-milliliter tube containing 10 milliliters of supplemented RPMI medium. Them, centrifuge the cells at 500 G for five minutes at four degrees Celsius.Next, remove the supernatant and resuspend the cells in 15 milliliters of supplemented RPMI medium. Then, transfer the cells to a T-75 culture flask and incubate them overnight with humidification. The next day, transfer the floating cells with a sterile 25-milliliter pipette to a 50-milliliter tube.Then, count the living cells using trypan blue exclusion. Next, isolate the CD4 positive cells from 10 million living, non-adherent PBMCs using a microbead separation column. Once the cells are isolated, plate and activate the T cells in Th1 and Th2 polarizing conditions according to the text protocol, and then isolate their nuclei.To being, prepare fresh lysis buffer and keep it chilled on ice. Also, in preparation for the transposition reaction, warm a thermal shaker to 37 degrees Celsius. Next, load a half million T cells into 1.5-milliliter microtubes and spin them down at 500 Gs for five minutes at four degrees Celsius.Next, wash the cells with one milliliter of cold PBS and repeat the spin cycle, but now suspend the cells in one milliliter of cold lysis buffer. Mix the cells with gentle pipetting to prevent the nuclei from being disrupted too much. Then, quickly take 10-microliter aliquots to observe the fraction of cells'lyse.Be sure to keep the cells cold and work quickly. Within five minutes, proceed with the transposition reaction. To begin, transfer 100, 000 nuclei to a 1.5-milliliter microtube and spin down the sample at 500 Gs for 10 minutes in a cold centrifuge.Then, gently remove the supernatant. Next, add the transposition reaction components to the nuclei. Then, resuspend the nuclei with gentle pipetting and incubate the nuclei on the thermal shaker at 500 RMP for 30 minutes to complete the transposition reaction.Next, perform a DNA cleanup step and elute the DNA fragments in 20 microliters of Tris hydrochloride. Then, use a PCR to make an initial amplification of the ATAC-sequencing libraries. Next, assess the optimal number of additional cycles needed for the final amplification by quantitative PCR.From the measured results, determine the number of cycles needed for the final PCR amplification of the ATAC-sequencing libraries. Then, perform the final amplification. Setting up an optimal size of DNA library fragments can improve the next generation sequencing.First, warm up the magnetic beads to room temperature and prepare fresh 70%ethanol in nuclease-free water. Next, add nuclease-free water to the ATAC-sequencing libraries to a volume of 100 microliters. Then, add 50 microliters of resuspended DNA binding magnetic beads.Pipette the beads into the DNA at least 10 times and wait five minutes. If any drops of beads get stuck on the tube wall or lid, briefly spin the tube. Now, place the sample next to the magnet for two minutes, and then transfer the supernatant to a new microtube.Measure the volume of the collection and add a 70%volume of magnetic bead suspension. Mix the beads in as before and let them incubate for five minutes before proceeding. Then, separate the beads using the magnet for two minutes and now discard the supernatant.While still against the magnet, add 200 microliters of 70%ethanol to the beads. Wait 30 seconds then discard the ethanol and repeat the ethanol wash once more. Finish the washes by completely removing the remaining ethanol and letting the beads air-dry for five minutes on the magnet.If necessary, briefly spin the tube and aspirate out any visible ethanol with a 10-microliter pipette. Now, remove the tube from the magnet and add 22 microliters of Tris hydrochloride. After two minutes, return the tube to the magnetic stand and let the solution clear.Then, collect 20 microliters of eluate containing the prepared library and store it at minus 20 degrees Celsius. This protocol produces an ATAC-sequencing libraries that are typically three to 20 nanograms per microliter. When run on a system for DNA integrity analysis, they have a ladder-like appearance.Amongst others, another important quality control is the enrichment of the positive controls. Relative to the negative controls, primer pair three should be enriched at least 25-fold, and primer pair four should be enriched at least 75-fold. After an initial 10 million reads, if the sample passes all crucial parameters in FastQC Report file, and 1, 000 to 2, 000 peaks are obtained from peak calling, then the library can be sequenced more deeply for greater than 30 million reads.From libraries meeting the quality standards, only six to 20%of the reads map to the mitochondrial genome. The remaining unique reads map to the reference human genome and seven to 12%are within the ATAC-sequencing peaks. After watching this video, you should have a good understanding of how to make ATAC-seq libraries, submit and furnish ration sequencing and to analyze the obtained data.Once mastered, the ATAC-seq library preparation can be done in one or two working days. While attempting this procedure, it is important to remember that you will maybe need to first optimize some steps like the number of the cells and the composition of the lysis buffer in a nuclei isolation step. Following this procedure, other methods like chromatin immunoprecipitation can be performed in order to answer additional questions like which transcription factor binds to the open chromatin regions in the examined samples?

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