See Protocol Exchange17 or Supplementary Protocols 1 and 2 for
a detailed protocol. Cells grown in tissue culture were pretreated with 200 U/ml
DNase (Worthington) for 30 min at 37 °C to remove free-floating DNA and
to digest DNA from dead cells. This medium was then washed out, and the cells
were resuspended in cold PBS. For primary human T cells, cells were sorted using
a Becton Dickinson FACS Aria II instrument based on the expression of CD45, CD3,
and CD4, as described previously11 (link). After the cells were counted, 50,000 cells were
resuspended in 1 ml of cold ATAC-seq resuspension buffer (RSB; 10 mM Tris-HCl pH
7.4, 10 mM NaCl, and 3 mM MgCl2 in water). Cells were centrifuged at
500 r.c.f. for 5 min in a pre-chilled (4 °C) fixed-angle centrifuge.
After centrifugation, 900 μl of supernatant was aspirated, which left
100 μl of supernatant. This remaining 100 μl of supernatant was
carefully aspirated by pipetting with a P200 pipette tip to avoid the cell
pellet. Cell pellets were then resuspended in 50 μl of ATAC-seq RSB
containing 0.1% NP40, 0.1% Tween-20, and 0.01% digitonin
by pipetting up and down three times. This cell lysis reaction was incubated on
ice for 3 min. After lysis, 1 ml of ATAC-seq RSB containing 0.1%
Tween-20 (without NP40 or digitonin) was added, and the tubes were inverted to
mix. Nuclei were then centrifuged for 10 min at 500 r.c.f. in a pre-chilled (4
°C) fixed-angle centrifuge. Supernatant was removed with two pipetting
steps, as described before, and nuclei were resuspended in 50 μl of
transposition mix (25 μl 2× TD buffer (recipe inSupplementary Protocol 1 ) 2.5
μl transposase26 (link) (100
nM final), 16.5 μl PBS, 0.5 μl 1% digitonin, 0.5
μl 10% Tween-20, and 5 μl water) by pipetting up and
down six times. Transposition reactions were incubated at 37 °C for 30
min in a thermomixer with shaking at 1,000 r.p.m. Reactions were cleaned up with
Zymo DNA Clean and Concentrator 5 columns. The remainder of the ATAC-seq library
preparation was performed as described previously18 . All libraries were amplified with a
target concentration of 20 μl at 4 nM, which is equivalent to 80
femtomoles of product. Minor protocol modifications were used for Omni-ATAC in
frozen tissues and in limiting cell numbers. These modifications are outlined in
the corresponding Online Methods sections.
a detailed protocol. Cells grown in tissue culture were pretreated with 200 U/ml
DNase (Worthington) for 30 min at 37 °C to remove free-floating DNA and
to digest DNA from dead cells. This medium was then washed out, and the cells
were resuspended in cold PBS. For primary human T cells, cells were sorted using
a Becton Dickinson FACS Aria II instrument based on the expression of CD45, CD3,
and CD4, as described previously11 (link). After the cells were counted, 50,000 cells were
resuspended in 1 ml of cold ATAC-seq resuspension buffer (RSB; 10 mM Tris-HCl pH
7.4, 10 mM NaCl, and 3 mM MgCl2 in water). Cells were centrifuged at
500 r.c.f. for 5 min in a pre-chilled (4 °C) fixed-angle centrifuge.
After centrifugation, 900 μl of supernatant was aspirated, which left
100 μl of supernatant. This remaining 100 μl of supernatant was
carefully aspirated by pipetting with a P200 pipette tip to avoid the cell
pellet. Cell pellets were then resuspended in 50 μl of ATAC-seq RSB
containing 0.1% NP40, 0.1% Tween-20, and 0.01% digitonin
by pipetting up and down three times. This cell lysis reaction was incubated on
ice for 3 min. After lysis, 1 ml of ATAC-seq RSB containing 0.1%
Tween-20 (without NP40 or digitonin) was added, and the tubes were inverted to
mix. Nuclei were then centrifuged for 10 min at 500 r.c.f. in a pre-chilled (4
°C) fixed-angle centrifuge. Supernatant was removed with two pipetting
steps, as described before, and nuclei were resuspended in 50 μl of
transposition mix (25 μl 2× TD buffer (recipe in
μl transposase26 (link) (100
nM final), 16.5 μl PBS, 0.5 μl 1% digitonin, 0.5
μl 10% Tween-20, and 5 μl water) by pipetting up and
down six times. Transposition reactions were incubated at 37 °C for 30
min in a thermomixer with shaking at 1,000 r.p.m. Reactions were cleaned up with
Zymo DNA Clean and Concentrator 5 columns. The remainder of the ATAC-seq library
preparation was performed as described previously18 . All libraries were amplified with a
target concentration of 20 μl at 4 nM, which is equivalent to 80
femtomoles of product. Minor protocol modifications were used for Omni-ATAC in
frozen tissues and in limiting cell numbers. These modifications are outlined in
the corresponding Online Methods sections.
