Mar 19, 2022
Tagmentation and library generation for human placental bulk ATACseq
- 1University of California, San Diego
Protocol Citation: Scott Lindsay-Hewett 2022. Tagmentation and library generation for human placental bulk ATACseq. protocols.io https://dx.doi.org/10.17504/protocols.io.261gen52jg47/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: March 19, 2022
Last Modified: March 19, 2022
Protocol Integer ID: 59630
Abstract
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.
Materials
Equipment:
Eppendorf ThermoMixer
Thermal cycler that will accommodate 96-well plate
Pipettes (including multichannel)
Magnetic stand
Qubit fluorometer
Agilent bioanalyzer
Supplies/reagents:
AMPure XP beads (Beckman Coulter, cat # A63881)
200 proof ethanol
Nuclease-free water
Tagment DNA TDE1 Enzyme (Illumina, cat # 20034197)
MinElute PCR Purification Kit (Qiagen, cat # 28004)
Sodium Acetate Solution, 3M, pH 5.2 (Fisher, cat # FERR1181)
IDT for Illumina Nextera DNA UD Index set (Illumina)
NEBNext High-Fidelity 2X PCR Master Mix (VWR, cat # 102500-096)
LoBind Eppendorf tubes (Eppendorf, cat # 022431021)
Qubit dsDNA HS Assay kit (Fisher, cat # Q32854)
Agilent High Sensitivity DNA Kit (Agilent, cat # 5067-4626)
Preparation
Preparation
Pre-heat Eppendorf ThermoMixer to 37 °C .
Bring AMPure XP beads to room temperature.
Prepare 70% ethanol using 200 proof ethanol and nuclease-free water.
Tagmentation and purification
Tagmentation and purification
Add 0.5 µL Tagment DNA TDE1 Enzyme to 50,000 freshly prepared nuclei (5,000 nuclei/ul in tagmentation buffer), and mix by pipetting gently.
Incubate in Eppendorf ThermoMixer 500 rpm, 37°C, 00:30:00 .
After completion, add 100 µL PB buffer (Qiagen) and 5 µL 3M Na-acetate, pH 5.2 . Purify using MinElute PCR Purification Kit and elute in 10 µL Buffer EB (Qiagen) .
Note
This is a safe stopping point. Store tagmented DNA at -20 °C until all samples are ready for library construction.
Amplification and indexing
Amplification and indexing
7m 10s
7m 10s
Thaw index adapter plate at room temperature, and spin briefly before use. Assemble PCR reactions as follows:
10 µL tagmented DNA
25 µL NEBNext High-Fidelity 2X PCR Master Mix
10 µL IDT for Illumina Nextera DNA UDIs
5 µL nuclease-free water
Note
Each well of the Illumina Nextera DNA UDI 96-well plate is single use and contains a little over 10 ul pre-paired i5/i7 index adapters. The Nextera adapters add 140 bp to the libraries.
Perform a limited-cycle amplification of the tagmented DNA:
Step 1: 72 °C 00:05:00
Step 2: 98 °C 00:00:30
Step 3: 98 °C 00:00:10
Step 4: 63 °C 00:00:30
Step 5: 72 °C 00:01:00
[Repeat steps 3-5 for a total of 8 cycles]
Step 6: 12 °C hold
7m 10s
After completion, add 250 µL PB buffer and 10 µL 3M Na-acetate, pH 5.2 . Purify using MinElute PCR Purification Kit and elute in 20 µL Buffer EB .
Size selection
Size selection
10m
10m
Add 180 µL Buffer EB and 110 µL AMPure XP beads (0.55x sample volume) and mix by pipetting. Incubate Room temperature 00:05:00 .
5m
Separate on magnetic stand, and transfer 300 µL supernatant to a new tube.
Add 250 µL AMPure XP beads (1.8x sample volume) and mix by pipetting.
Incubate Room temperature 00:05:00 .
5m
Separate on magnetic stand and wash beads twice with 200 µL 70% ethanol .
Resuspend beads in 20 µL Buffer EB by pipetting.
Separate on magnetic stand and transfer 17 µL of size-selected library to a new tube.
Quality control
Quality control
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.
Note the nucleosomal laddering pattern, which corresponds to nucleosome-free, mononucleosomal, dinucleosomal, and trinucleosomal fragments enriched at ~200, 350, 550, 750bp, respectively.
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.
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.
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.
Note
Tip: set a region from 200bp to 1000bp in the region table tab (fragments over ~1kb don't cluster efficiently on the flow cell) and the Agilent software will calculate the average fragment size.
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.