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ATAC-seq Protocol


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ATAC-seq Protocol

ATAC seq Protocol

Cellpreparation

1. 50,000 cells are counted and harvested, then cells are centrifuged 5 min at 500g, 4℃.

2. Remove and discard supernatant. Wash cells with 50 uL cold PBS buffer and centrifuge 5 min at 500g, 4℃.

3. Remove and discard supernatant. Resuspend the cell pellet with 50 uL cold lysis buffer centrifuge 10 min at 500g, 4℃.

4. Discard the supernatant, the Lyse cells will be generate a crude nuclei. Next, they need immediately continue to transposition reaction.

Construction of Transposome

5. To make the reaction system, combine the following:

The joint that connects with a ME sequence  1.0-1.5 uL
10×TPS buffer 2 uL
Robust Tn5 Transposase 1 uL
TE buffer Up to 20 uL

6. Incubate the mixture at 25℃ for 30 min.

7. The transposome can be immediately used to transposition reaction, or stored at -20℃ if necessary.

Tagmentation and purification

8. To make the transposition reaction mix, combine the following:

DNA to be fragmented ( from step 4) 100 ng
5×LM buffer 6 uL
Transposome 4 uL
Nuclease-free H2O Up to 30 uL

9. Incubate the transposition reaction mix at 37℃ for 2 hours or at 56℃ for 10-15 min.

10. Purify the fragmented and tagged DNA using a Creative Biogene PCR Purification Kit. Stored purified DNA at -20℃ if necessary.

PCR amplification and purification

11. Amplify transposed DNA fragments using a Creative Biogene PCR amplification Kit.

12. Purify the PCR amplified library using a Creative Biogene PCR Purification Kit.

Library sequencing

Sequence the library and correlate reads with open and closed chromatin. When sequencing ATAC-seq libraries, sequencing primers and reagents must be used. For nucleosome mapping, paired-end sequencing is preferred. Foe inferring differences in open chromatin with human samples,we generally use>50,000,000 mapped reads, and for transcription factor foot-printing, we use>200,000,000 mapped reads. The mitochondrial read fraction can vary between 10% and >50%, depending on cell type.

Notes

1. At the start of the experiment, cells must be at healthy living condition to ensure obtain more complete information in subsequent studies.

2. Cells should be lysed under mild conditions to ensure that the structure of the chromosomes is not destroyed.

3. After the treatment of Tn5 transposase, purify the fragmented is necessary, Otherwise it can produce false positives.

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