Tagmentation procedure for ATAC-seq of sorted GFP+ endothelial cells

Solutions to prepare

Buffer HB (0.2 um sterile filter/store at 4 °C)

0.25M sucrose

25 mM KCl

5 mM MgCl2

20 mM Tricine-KOH

pH 7.8

Lysis Buffer (prepare the day of the experiment)

Buffer HB with 0.1% (v/v) Igepal CA-630

1)    Centrifuge 50,000 cells for 5 minutes at 500 x g at 4 °C. Turn tube 180 degrees and repeat centrifugation for another 5 minutes. This ensures most cells are at the bottom of the tube and not stuck to sides.

2)    Remove supernatant carefully. It is very difficult to see a cell pellet. Gently pipet up and down to resuspend cells in 50 ul of cold lysis buffer. Centrifuge immediately for 10 minutes at 500 x g at 4 degrees centigrade. Rotate tube 180 degrees and repeat centrifugation for another 10 minutes.

3)    Remove supernatant carefully. Place nuclei pellet on ice and proceed to transposition reaction.

4)    Prepare transposition reaction mix:

a.     25 ul TD buffer (2x reaction buffer from Nextera kit)

b.     2.5 ul TDE1 (Nextera Tn5 Transposase from Nextera kit)

c.     22.5 ul nuclease-free water

5)    Resuspend nuclei pellet in transposition reaction mix.

6)    Incubate the reaction at 37 °C for 30 minutes.

7)    Purify DNA using Qiagen MinElute GelExtraction Kit. The solubilization Buffer QG gives better results than MinElute PCR purification kit. At this point, you can add the yellow Buffer QG and freeze sample at -80 °C or finish purification.

8)    Elute transposed DNA in 20 ul of elution buffer (Buffer EB). The original protocol calls for 10 ul elution, and they add 10 ul nuclease-free water in the library preparation PCR step. You can elute 20 ul and don’t add water for PCR.

9)    Can store DNA if necessary at -80 °C.

Library Preparation

The original protocol calls for a qPCR step to determine how many cycles to do. We find that 11 total cycles typically gives a decent library. See the original paper for primers to use.

1)    To amplify transposed DNA fragments, combine the following in a 0.2 ml PCR tube:

a.     20 ul transposed DNA

b.     2.5 ul 25 uM ATAC PCR Primer 1

c.     2.5 ul 25 uM ATAC Barcoded PCR primer 2

d.     25 ul NEBNext High-Fidelity 2x PCR Master Mix

2)    Thermal cycle as follows:

a.     1 cycle             5 min                   72 °C

        30 sec                  98 °C

                           11 cycles         10 sec                  98 °C

                                                    30 sec                  63 °C

                                                    1 min                   72 °C

                            Hold                Infinite                    4 °C

3)    Purify library using Agencourt AMPure XP beads. Elute from beads using 12 ul of Buffer EB from Qiagen MinElute kit and take 10 ul.

4)    Assess the library using Bioanalyzer or Fragment Analyzer. Our experience with ATAC-seq is for whatever reason some libraries do not give a good/recognizable laddering pattern but when the libraries are sequenced, the data look reasonable.