TITLE: Tagmentation and library generation for human placental bulk ATACseq
AUTHORS: Scott   Lindsay-Hewett

[DESCRIPTION]
This protocol describes the generation and amplification/indexing of tagmented libraries from freshly isolated nuclei for bulk ATACseq. It is adapted from Buenrostro et al., 2015, PMID: 25559105.

[STEPS]
SECTION: Preparation
1. Pre-heat Eppendorf ThermoMixer to 37 °C.

SECTION: Tagmentation and purification
4. Add 0.5 µL to 50,000 freshly prepared nuclei (5,000 nuclei/ul in tagmentation buffer), and mix by pipetting gently.

SECTION: Tagmentation and purification
5. Incubate in Eppendorf ThermoMixer  .
SECTION: Tagmentation and purification
6. After completion, add 100 µL and 5 µL. Purify using MinElute PCR Purification Kit and elute in 10 µL.
 


SECTION: Amplification and indexing
7. Thaw index adapter plate at room temperature, and spin briefly before use. Assemble PCR reactions as follows:

10 µL 
25 µL 
10 µL 
5 µL 
 


SECTION: Amplification and indexing
8. Perform a limited-cycle amplification of the tagmented DNA:

Step 1:    72 °C 5 min 
Step 2:    98 °C 30 s 
Step 3:    98 °C 10 s 
Step 4:    63 °C 30 s 
Step 5:    72 °C 1 min 

[Repeat steps 3-5 for a total of 8 cycles]

Step 6:    12 °C 

SECTION: Amplification and indexing
9. After completion, add 250 µL and 10 µL. Purify using MinElute PCR Purification Kit and elute in 20 µL.
SECTION: Size selection
10. Add 180 µL and 110 µL (0.55x sample volume) and mix by pipetting. Incubate Room temperature 5 min. 

SECTION: Size selection
11. Separate on magnetic stand, and transfer 300 µL supernatant to a new tube.

SECTION: Size selection
12. Add 250 µL (1.8x sample volume) and mix by pipetting. 
Incubate Room temperature 5 min. 
SECTION: Size selection
13. Separate on magnetic stand and wash beads twice with 200 µL.

SECTION: Preparation
2. Bring AMPure XP beads to room temperature.
SECTION: Preparation
3. Prepare 70% ethanol using 200 proof ethanol and nuclease-free water.
SECTION: Size selection
14. Resuspend beads in 20 µL by pipetting.

SECTION: Size selection
15. Separate on magnetic stand and transfer 17 µL of size-selected library to a new tube.

SECTION: Quality control
16. Quantitate libraries using the Qubit DNA High Sensitivity Assay, and check library distribution by running the DNA High Sensitivity Assay on an Agilent Bioanalyzer. A typical trace is shown below. 
  
Pay attention to the presence of any primer dimers, which will appear at ~50-80 bp. These can occupy space on the flow cell and reduce the number of useful reads generated from a sequencing run. If primer dimers are present (even at low levels), perform another 1.8x cleanup using AMPure XP beads.

SECTION: Quality control
17. If multiplexing samples, first perform a balancing run to ensure equal representation of all samples in the pool. For the balancing run, prepare an "equal volume" pool by combining 2 µL each library together. Run the pool on a MiSeq instrument using a MiSeq Reagent Kit Nano. Based on the proportion of reads assigned to each index during the Nano run, prepare a balanced pool that will yield an equal read depth for all samples in the pool.

SECTION: Quality control
18. Prior to submitting for sequencing, quantitate the pool using the Qubit High Sensitivity DNA assay. Determine the average fragment size for each library from the Bioanalyzer traces.
 

Determine the average fragment size in the balanced pool, and use the following formula to determine the nM concentration:

  
Submit the pool to your sequencing facility, noting the nM concentration.


For HuBMAP bulk ATAC-seq samples, the multiplexed pool was sequenced on a NovaSeq 6000 S4 lane using a 100bp paired-end run configuration.