TITLE: ChIP-seq Library preparation
AUTHORS: Vasso Makrantoni, Daniel Robertson, Adele L. Marston

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">A plethora of biological processes like gene transcription, DNA replication, DNA recombination, and chromosome segregation are mediated through protein–DNA interactions. A powerful method for investigating proteins within a native chromatin environment in the cell is chromatin immunoprecipitation (ChIP). Combined with the recent technological advancement in next generation sequencing, the ChIP assay can map the exact binding sites of a protein of interest across the entire genome. Here we describe a-step-by step protocol for ChIP followed by library preparation for ChIP-seq from yeast cells.</div></div>

[STEPS]
?. [ChIP-seq Library preparation]
There are commercially available kits for generating DNA libraries but it is relatively straightforward and cost effective to create libraries using standard molecular biology reagents and custom oligonucleotides. This protocol can be completed within 1 day, and it comprises five distinct steps: blunting reaction; dA-Tailing to the 3′ end of the DNA fragments; adapter ligation to the DNA fragments; PCR for enrichment of adapter modified DNA fragments; and library size selection. Finally, given the high cost of ChIP-seq runs and the time-intensive bioinformatics analysis and data validation, it is essential that the quality and the concentration of the libraries is validated by an Agilent Bioanalyzer prior to sequencing.
?. [DNA End-Repair]
Elute in  and take  to a new DNA LoBind Eppendorf.
[ddH2O]
27.7 µl
?. [“A”-Tailing Reaction]
Proceed immediately to adapter ligation reaction (next step).
?. [PCR Amplification Reaction]
Amplify DNA with the following PCR program:   ABC1TemperatureTimeCycles298 °C30 sec398 °C10 sec12 -18 cycles465 °C30 sec572 °C30 sec672 °C5 min
ABC1TemperatureTimeCycles298 °C30 sec398 °C10 sec12 -18 cycles465 °C30 sec572 °C30 sec672 °C5 min
When PCR for library amplification is performed, minimal cycling is desirable. The fewer number of PCR cycles used to amplify libraries, the less biased the resulting libraries will be for the products that are more efficiently amplified. Overamplification can result in daisy-chains of fragments that can be visualized as a higher molecular weight peak on the Bioanalyzer. If the library amplification fails, more DNA template can be used. For IP samples use up to 20 μl of template DNA.
?. [Double-Sided AMPure Selection and Library Elution]
The final concentration of the library to load on the flow cell is 1.5 pM with an Input–IP ratio 15%:85%. Perform paired-end sequencing with 76 bp—76 bp for Read 1 and Read 2.
Several different next generation sequencers are available; for this protocol we use the Illumina platform. While platforms vary by target sequence length, accuracy and cost all give reproducibly comparable results. In order to perform experiments in a cost-effective manner, multiplexing can be used, that is, multiple ChIP-seq libraries, each carrying a different barcode to identify different samples can be sequenced together on a single flow cell of MiniSeq or lane of a Hiseq. The output of the Illumina MiniSeq is ~25 M clusters. We typically, sequence 8–10 barcoded uncalibrated samples in a single pooled library or 4–5 barcoded calibrated samples in a single pooled library. Typically one ChIP library generates six to ten million reads [23,24]. However, the above is subject on the level of enrichment of the protein of interest and the resolution required.
?. [DNA End-Repair]
Perform the blunting reaction using the following recipe: if making multiple libraries prepare a master mix of buffer, dNTPs, and enzyme, and then aliquot to the required ChIP purified DNA. The final volume should be 50 μl.(a) –  (ideally 2 ng)(b) (c) (d)
1 ng
[ChIP DNA]
[10× blunting buffer]
[1 mM dNTPs]
[blunting enzyme]
For library preparation we recommend to use only filter-tips and 1.5 ml DNA LoBind tubes.
?. [DNA End-Repair]
Incubate at  for .
65 Room temperature
?. [DNA End-Repair]
Perform a 1.6:1 AMPure XP selection by adding  to the  blunting reaction (see protocol "AMPure Purification Protocol").
[AMPure beads]
[blunting reaction]
?. [“A”-Tailing Reaction]
Use end-repaired DNA (from step 5) to perform “A”-tailing reaction using the following recipe: if making multiple libraries prepare a master mix of buffer, dATP, and enzyme, and then aliquot to the required end-repaired DNA. The final volume should be 30 μl.(a) (b) (c) (d)
[end-repaired DNA]
[10× NEB buffer 2]
[10 mM dATP]
[Klenow 3′ to 5′ exo minus (5 U/μl)]
?. [“A”-Tailing Reaction]
Incubate at  for .
37 °C
?. [“A”-Tailing Reaction]
Heat-inactivate Klenow enzyme at  for .
75 °C
?. [“A”-Tailing Reaction]
Place reaction  for .
on ice
?. [Adapter Ligation Reaction]
Use dA-tailed DNA (from previous step) to perform the adapter ligation reaction using the following recipe: the final volume should be 70 μl. Use different barcoded adapters for each Input and IP sample.(a) (b) (c)   [15](d)
[“A”-tailed DNA]
[2× Quick Ligase buffer]
[0.5 μM Adapters]
[Quick T4 DNA ligase (2000 U/μl)]
Always keep adapters on ice. The quantity of adapters stated here is recommended for 2 ng of DNA; however, the amount of adapters should be proportional to the amount of DNA used. Optimal concentration of adapters used is essential. High concentration can lead to adapter contamination in the final library, which can be visualized on the Bioanalyzer (Fig.2b). We use for this protocol the NEXTflex DNA Barcodes—12 (Bioo Scientific; #NOVA-514102). The NEXTflex DNA Barcodes utilize an indexed adapter containing a 6 nt unique sequence. Details can be found in manufacturer’s manual.
?. [Adapter Ligation Reaction]
Incubate at  for .
0 Room temperature
?. [Adapter Ligation Reaction]
Perform a 1:1 AMPure selection by adding  to the  (see protocol "AMPure Purification Protocol").
[AMPure XP beads]
[adapter ligation reaction ]
?. [Adapter Ligation Reaction]
Elute in . Transfer  to a new DNA LoBind Eppendorf.
[ddH2O]
50 µl
DNA fragments over 100 bp will bind to beads and be eluted.
?. [Adapter Ligation Reaction]
Perform another 1:1 AMPure selection by adding  to the  from the previous step.
[AMPure beads]
[elution fraction]
?. [Adapter Ligation Reaction]
Elute in  and take up  to a new DNA LoBind Eppendorf.
[ddH2O]
30 µl
DNA fragments above 100/200 bp will bind to beads and be eluted.
?. [PCR Amplification Reaction]
Transfer  per sample (previous step) to a 0.2 ml PCR tube and set up the following PCR reaction  in a final volume of 50 μl.(a) (b) (c) (d) (e) (f) (g)
[supernatant ]
on ice
[ adapter-ligated DNA]
[5× Phusion HF buffer]
[2.5 mM dNTPs]
[12.5 μM Primer mix]
[DMSO]
[Phusion polymerase]
[ddH2O]
?. [Double-Sided AMPure Selection and Library Elution]
Perform a 0.65×:1 AMPure selection. To the  (previous step) add , resuspend by pipetting and leave at  for  to bind.Place 0.2 μl PCR tubes in a DynaMag-PCR magnet for .
[PCR reaction]
[AMPure XP beads]
0 Room temperature
?. [Double-Sided AMPure Selection and Library Elution]
KEEP THE SUPERNATANT, this will contain fragments  add  (adjust bead volume for smaller supernatant volumes) and perform AMPure selection according to steps in protocol "AMPure Purification Protocol".
[supernatant]
[Ampure beads]
DNA fragments of 100–250 bp will bind to the beads.
?. [Double-Sided AMPure Selection and Library Elution]
Elute in  and pipet  to a new DNA LoBind Eppendorf.
[EB (Qiagen purification kit) or ultrapure ddH2O]
50 µl
?. [Double-Sided AMPure Selection and Library Elution]
Perform another 1:1 AMPure selection by adding   to the  from the previous step.
[AMPure beads]
[elution fraction]
DNA fragments above 100/200 bp will bind to the beads.
?. [Double-Sided AMPure Selection and Library Elution]
Elute in  and transfer  to a new LoBind Eppendorf.
[ EB buffer (Qiagen purification kit)]
30 µl
?. [Double-Sided AMPure Selection and Library Elution]
Determine DNA library concentration by Qubit HS kit (use ).
2 µl
?. [Double-Sided AMPure Selection and Library Elution]
Run the library on a Bioanalyzer to determine average fragment size and general purity. Use a High Sensitivity DNA Kit (Agilent Technologies) as per manufacturer’s instructions. Fragments of the sequencing library should have a size range of 150–300 bp (Fig.2b, upper panel). If not pure, that is, adapter dimers are visible (Fig.2b, lower panel), perform 1:1 AMPure purification to remove small adapter dimers.
?. [Double-Sided AMPure Selection and Library Elution]
Libraries are now ready for sequencing using a sequencing platform of choice.