ATAC-seq protocol

Materials:

10mM TrisHCl

5mM MgCl₂

DMF (N,N-Dimethylformamide for molecular biology, ≥99%; Sigma CAS number: 68-12-2)

NP-40 (Nonidet P40; catalog number: 19628; CAS number: 9002-93-1)

Tn5 Transposase

Buffer ERC (QIAGEN Enzyme reaction cleanup buffer)

QIAGEN MinElute column

NEBNext® High-Fidelity 2X PCR Master Mix

Illumina primers (Nextera DNA Library Preparation Kit)

AMPure beads (AMPure XP for PCR Purification Cleanup and Size Selection; Beckman Coulter, A63880)

Agilent D5000 ScreenTape, Sample Buffer and Ladder (Catalog Code: 5067-5588, 5067-5589)

Prepare cells: 

1000-5000 cells per reaction (1000 cells minimum), dissociate to single cells

A. Add 20ul Lysis Buffer to each tube

Incubate on ice for 15min

B. Add 30ul Reaction Buffer to each tube

Incubate at 37°C for 20 min

C. Add 300ul Buffer ERC (QIAGEN Enzyme reaction cleanup buffer) to stop reaction

ATAC – build library

1. Extract DNA

Transfer (Tn5+ERC) solution to QIAGEN MinElute column

Spin for 1min

Add 700ul Buffer PE, spin

Wash 2x

Spin empty column

Add 11.5ul H₂O, spin

Add another 11.5ul, spin

= obtain total 23ul extracted DNA

2. Pre-PCR

PCR reaction:

3. qPCR (to decide how many more cycles to use for library construction)

Take 5ul from pre-PCR reaction product

      + 5ul NEBnext mix

      + 5ul H2O

total = 15ul

qPCR reaction:

15ul reaction volume

select run 20min

4. library

AMPure beads 

45ul PCR product to each tube + 45ul beads mixture 

wait 5 min, spin down, put on rack, wait 2 min

80% alcohol wash x2

20ul elution buffer (EB)

vortex, leave RT 2 min, spin, put back to rack

transfer liquid to new tube

5. Tape station prepare (D5000 ScreenTape)

Add 1.5 ul Agilent High Sensitivity D5000 sample buffer green label to each tube

Add 1.5 ul D5000 ladder to 1^st tube as control

Add 1.5 ul sample (eluted DNA) to other tubes

Note: 

(1) Tn5 transposase used in this protocol was homemade (Picelli, et al., 2014, Sandberg lab). 

(2) No need for nuclei isolation, and this protocol will not generate more than 5% mitochondrial reads after sequencing.

(3) Recommended cell number for this protocol is 500 - 3000.

(4) Please check the attachment for a detailed version. 

(5) We will publish the validation of this protocol within one of our manuscripts soon.

Protocol:

(1) Fluorescent protein expressing cells from zebrafish embryos were FACS purified into 300uL PBS with 2% FBS.

(2) Centrifuge at 700 xg for 15 min, gently remove most of the supernatant, leaving no more than 10uL PBS in the tube.

(3) Add 20uL lysis buffer directly to the cells, pipette 5 times to lyse the cell membrane. (lysis buffer: 10mM Tris-HCl pH 7.4, 5mM MgCl2, 10% DMF, 0.2% NP-40)

(4) Add 30uL reaction buffer directly to the cell lysis, vortex and incubate at 37 C for 20min. (reaction buffer: 10mM Tris-HCl pH 7.4; 5mM MgCl2, 10% DMF, 1uL homemade transposase)

(5) Stop reaction by adding 300uL ERC buffer from Qiagen MiniElute kit. 

(6) Build libraries (ATACseq protocol from Greenleaf lab at Stanford). 

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