ATAC Sequencing Protocol

1. To use for Lysis Buffer and Wash Buffer, can be stored at room temperature long-term.

500 μl 1M Tris-HCl, pH 7.5 (final 10 mM)100 μl 5 M NaCl (final 10 mM)150 μl 1 M MgCl2 (final 3 mM) 49.25 ml nuclease-free H_²O

50mL

1. Centrifuge 100,000 cells to pellet [NOTE: The person who is sorting and aliquoting cells will tell you speed/time, to centrifuge usually this is 8,000 xg for 4 minutes] NOTE : The person who is sorting and aliquoting cells will tell you speed/time, to centrifuge usually this is 8,000 xg for 4 minutes], and then discard supernatant.

2. Add 100µL cold Lysis Buffer, pipet up and down 3x gently to resuspend cells. [NOTE: Lysis buffer volume should be scaled to the number of cells; e.g. if you only have 50,000 cells, add NOTE : Lysis buffer volume should be scaled to the number of cells; e.g. if you only have 50,000 cells, add 50µL cold Lysis Buffer]

97 μl   Resuspension Buffer1 μl   10% NP-40 (final 0.1% v/v)1 μl   10% Tween-20 (final 0.1% v/v) 1 μl   1% Digitonin (final 0.01% v/v)100µL 3. Incubate on ice x3 minutes. 4. Add 1mL Wash Buffer, invert tube 3 times gently.990 μl Resuspension Buffer 10 μl 10% Tween-20 (final 0.1% v/v)1mL

5. Centrifuge at 500 xg for 10 minutes at 4°C .

6. Discard supernatant (cytoplasm), keep pellet (nuclei).

1. While cells are centrifuging, make transposition reaction mix, using the Nextera DNA Library Prep Kit.  [NOTE: Transposition reaction mix volume should be scaled to the number of cells; e.g. if you only have 50,000 cells, add NOTE : Transposition reaction mix volume should be scaled to the number of cells; e.g. if you only have 50,000 cells, add 50µL transposition mix]

50 μl  2X TD Buffer (Tagment DNA Buffer)

33 μl  1X PBS

1 μl  10% Tween-20 (final 0.1% v/v)

1 μl  1% Digitonin (final 0.01% v/v)

5 μl  Tn5 Transposase (Tagment DNA Enzyme 1)

10 μl nuclease-free H_²O

100µL

2. Add transposition reaction mix to pellet, pipet up and down 6x gently to resuspend nuclei.

3 . Incubate at 37°C for 30 minutes on thermomixer at 1,000 rpm.

1. Isolate DNA using Qiagen MinElute Reaction Cleanup Kit.

2 . Elute DNA in 10 μl EB (Elution Buffer).

Note:

OK to store DNA at -20°C at this point.

1. Combine the following in a PCR tube for each sample:

10 μl    purified transposed DNA

10 μl    nuclease-free H_²O

2.5 μl   Ad1_noMX primer (25 μM)

2.5 μl   Ad2.* indexing primer (25 μM)

25 μl     NEBNext High-Fidelity 2X PCR Master Mix

50µL

2. Amplify samples in PCR machine with following program:

72°C 5 minutes     x5 cycles

98°C 30 seconds   x5 cycles

98°C 10 seconds   x5 cycles

63°C 30 seconds   x5 cycles

72°C 1 minute       x5 cycles

3. Remove tubes from PCR machine and use 5µL of each partially-amplified library to perform qPCR to determine how many additional PCR cycles are needed.  The goal is to stop amplification well prior to saturation to avoid variation among samples due to PCR bias.

5 μl      partially-amplified library

3.85 μl nuclease-free H_²O

0.5 μl   Ad1_noMX primer (25 μM)

0.5 μl   Ad2.* indexing primer (25 μM)

0.15 μl 100X SYBR Green I

5 μl       NEBNext High-Fidelity 2X PCR Master Mix

15µL

4. Perform qPCR using following program:

98°C     30 seconds x20 cycles

98°C     10 seconds x20 cycles

63°C     30 seconds x20 cycles

72°C     1 minute      x20 cycles

5. **qPCR Plate setup:

a. Well type: unknown

b. Collect fluorescence data for ROX and FAM

c. Reference dye: ROX

6. **qPCR Thermal Profile setup:

a. Pre-Melt/RT Segment: 1 plateau



b. Amplification Segment: normal 3 skip

c. Dissociation/Melt segment: uncheck this box

7. Plot R vs Cycle Number.  Calculate the number of additional PCR cycles needed for each sample, by determining the number of cycles needed to reach 1/3 of the maximum R.

8 . Continue PCR on remaining 45µL of each partially-amplified library for the appropriate number (N) of cycles:

98°C     30 seconds N cycles

63°C     30 seconds N cycles

72°C     1 minute      N cycles

1. Warm AMPure XP beads to room temperature, and vortex for 15 seconds to resuspend.

2. For double-sided bead purification (to remove primer dimers and large >1,000 bp fragments):

a. Transfer each PCR sample to an epi tube, add 0.5X volume (22.5µL ) AMPure XP beads, pipet up and down 10x to mix thoroughly.

b. Incubate at room temperature for 10 minutes.

c. Place epi tubes in magnetic rack for 5 minutes.

d. Transfer supernatant to new epi tube.

e. Add 1.3X original volume (58.5µL ) AMPure XP beads, pipet up and down 10x to mix thoroughly.  (This results in a final 1.8X bead buffer:sample ratio.)

f. Incubate at room temperature for 10 minutes.

g. Place epi tubes in magnetic rack for 5 minutes.

h. Discard supernatant.

i. Wash beads with 200µL 80% EtOH (freshly made), pipet EtOH over beads 10x, then discard EtOH.

j. Leave tube on magnetic rack with cap open for 10 minutes.

k. Ensure all EtOH is removed.

l. Resuspend beads in 20µL nuclease-free H_²O, pipet up and down 10x to mix thoroughly.

m. Incubate at room temperature for 2 minutes, then quickly spin epi tube down

n. Place epi tube in magnetic rack for 1-5 minutes.

o. Transfer supernatant to new epi tube.

3 . Store purified libraries at -20°C .

1. Add 1µL of each library to 3µLnuclease-free H_²O (to make 1:4 dilution).

2. Run 1µL of each diluted library on an Agilent High Sensitivity DNA Bioanalysis chip.

3. Use 1µL of each diluted library to measure DNA concentration by QuBit.

1. Use 50 bp paired-end (50PE) sequencing.

2. Goal is to obtain >50 million genomic reads per sample minimum to assess open vs closed chromatin regions, and >200 million genomic reads per sample to detect transcription factor binding sites.  Remember that many sequencing reads may map to contaminating mitochondrial DNA.

Table of PCR Primers (Illumina/Nextera i5 common adapter and i7 index adapters):