TITLE: A simple ATAC-seq protocol for population epigenetics
AUTHORS: Ronaldo De Carvalho Augusto, Aki MINODA, Christoph Grunau

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">We describe here a step-by-step protocol for the generation of sequence-ready libraries for the Assay for Transposase Accessible Chromatin (ATAC-seq) that provides a positive display of accessible, presumably euchromatic regions. The protocol is straightforward and can be used with small individuals such as daphnia and schistosome worms, and probably many other biological samples of comparable size. This makes the method suitable for population epigenomics studies. This protocol is based on the protocol from Corces et al. (2016), Buenrostro et al. (2015) and Nextera DNA Library Prep Kit (2017) with some modifications. Controls must be done without organisms as input.</div></div>

[STEPS]
?. Step 1: Daphnia/Schistosoma samplingSet Eppendorf ThermoMixer with agitation to 37°C.1. Cut a 1 mL pipette tip to create larger opening2. Catch a single daphnia with pipette3. Take a microphotograph4. Remove all water by pipetting with 100 μL tip;orPerfuse S.mansoni worms and take single worm as dry as possible with forceps5. Wash once with 50 μL of cold 1x PBS buffer andremove all supernatant by pipetting, being careful not to remove your sample;6. Add to each sample25 μl 2× TD buffer2.5 μl TDE10.5 μl 1% IGEPAL22 μl Nuclease-free waterThis gives 50 μL of transposase mixture for each sample.Pipette up and down 10 times to disrupt cells. Proceed immediately to step 2.
?. Step 2: Chromatin TagmentationThis step uses the Nextera Tn5 transposome to tagmentthe chromatin, which is a process that fragments the chromatin and tags the DNA with adapter sequences in asingle step. 1. Tagmentation reactions is incubated at 37°C for30 min in an Eppendorf ThermoMixer withagitation at 300 rpm.2. Tagmented chromatin is immediately purified using a QIAGEN MinElute Reaction Cleanup kit (#28204) or a QIAquick PCR Purification Kit (#28106), and purified DNA is eluted into 20 μl of elution buffer (10 mM Tris-HCl, pH 8)
?. Step 3: Library amplificationThis step amplifies the tagmented DNA using a limited cycle PCR program. PCR is carried out with a universal index Ad1 and an index (barcode) primer Ad2.*.Option 1 (for Promega GoTag G2): Combine the following in a PCR tube for each sample9.5 μl Nuclease-free MilliQ water20 μl Purified transposed DNA10 μl 5x GoTaq G2 buffer4 μl MgCl22.5 μl Universal Ad1_noMX primer (25μM)2.5 μl Specific Index primer Ad2.*,different for each sample (25μM)1 μl dNTPs (10 mM)0.5 μl GoTaq G2OrOption 2 (for NEB mix, more convenient but more expensive):20 μl Purified transposed DNA2.5 μl Universal Ad1_noMX primer (25μM)2.5 μl Specific Index primer Ad2.* ,different for each sample (25μM)25 μl NEBNext High-Fidelity 2X PCR Master MixIn both options the final volume is 50 μl.Pre-amplify samples in PCR machine with following program:Step                                 Temp     Duration     CyclesPre-Warming                   72°C         5 min             1Initial denaturation         98°C       30 sec             1Denaturation                    98°C      10 secAnnealing                         63°C      30 sec              5Extension                         72°C         1 minHOLD                                12°C         ∞ In order to reduce GC and size bias in PCR, the PCR reaction is monitored using qPCR to stop amplification priorsaturation.To run a qPCR side reaction, combine the following:5 μl PCR product of the initial preamplification reaction (keep the remaining 45 μl at 4°C)2.5 μl 5x GoTaq G2 buffer0.1 μl GoTaq 23.14 μl Nuclease-free MilliQ water0.25 μl Universal Ad1_noMX primer (25μM)0.25 μl Ad2.* indexing primer (25μM)1 μl MgCl20.25 μl dNTPs0.1 μl 100X SYBR ITotal 12.5 μlor (when using NEB mix)5 μl PCR product of the initial preamplification reaction (keep the remaining 45 μl at 4°C)4.41 μl Nuclease-free MilliQ water0.25 μl Ad1_noMX primer (25 μM)0.25 μl Ad2.* indexing primer (25 μM)0.09 μl 100X SYBR I5 μl NEBNext High-Fidelity 2X PCR Master MixTotal 15 μlAmplify samples in a qPCR machine with following program:Step                                 Temp     Duration     CyclesInitial denaturation         98°C        30 sec         1Denaturation                   98°C        10 secAnnealing                        63°C        30 sec         20Extension                        72°C         1 minHOLD                               12°C             ∞ To calculate the optimal additional number of cyclesneeded for the remaining 45 μl PCR, plot Relativefluorescence versus cycle number, and determine the cyclenumber that corresponds to one-third of the maximumfluorescent intensity (figure 1). In our experience, total ofamplification cycles should not exceed 21.Run the remaining 45 μl PCR reaction with the additional number of cycles and purify with a QIAGEN MinEluteReaction Cleanup kit (#28204) or a QIAquick PCR Purification Kit (#28106) or similar, and elute with 20 μl ofelution buffer (10 mM Tris-HCl, pH 8). Run it on a 1.5% agarose gel or a Bioanalyzer chip. You should see a ladder that corresponds to the nucleosomefree region and multiple nucleosome-size fragments (one nucleosome = about 150 bp). A single band at around 150bp indicates sample degradation or over-fragmentation.Ideally, five bands should be obtained, three bands are acceptable (figure 2).
?. Step 4: AMPure XP beads double side purification This step enriches for the nucleosome-free (~300 bp).Removing small fragments (primer dimers) is important for optimal sequencing. We have not systematically investigated if different purification procedures influence on the result.1. Transfer 45 μl to an Eppendorf tube (or use PCR tube directly). Add 22.5 μl (0.5X original volume, to remove large fragments) AMPure XP beads, pipet up and down 10 times to mix thoroughly.2. Incubate at room temperature for 10 minutes.3. Place tubes in magnetic rack for 5 minutes.4. Transfer supernatant to new tube.5. Add 58.5 μl (1.3X original volume, to remove small fragments) AMPure XP beads, pipet up and down 10 times to mix thoroughly6. Incubate at room temperature for 10 minutes.7. Place tubes in magnetic rack for 5 minutes.8. Discard supernatant.9. Wash beads with 200 μl 80% ethanol (freshly made), pipet ethanol over beads 10 times, then discard ethanol. Ensure all ethanol is removed.10. Leave tube on magnetic rack with cap open for 3 to maximum 10 minutes depending on ambient humidity. The beads should be ‘glowing’ but not wet. Be careful not to over-dry them, which will decrease elution efficiency.11. Resuspend beads in 20 μl nuclease-free water, pipet up and down 10 times to mix thoroughly.12. Place tube in magnetic rack for 1-5 minutes.13. Transfer supernatant to new tube.OR: use Diagenode IP-Star, size selection 320 bp.Store purified libraries at -20˚C.
?. Step 5: Libraries checkPerform size profiling on Agilent Bioanalyzer High Sensitivity DNA Assay. Expected profiles are shown in figure 3. Use Bioanalyzer profiles or KAPA library quantification kit to quantify libraries and proceed to sequencing.
?. [End matter]
NotesIt would certainly be good to have always the same number of fragments after Tn5 incubation. It could beenvisaged to incubate aliquots of each sample for 10, 20 and 30 min, and then use the sample from the incubationtime that provides a fixed, e.g. 3-nucleosome peaks for all samples.TD buffer can be prepared as follows: 20 mM Tris(hydroxymethyl)aminomethane; 10 mM MgCl2; 20%(vol/vol) dimethylformamide (Wang et al. 2013)Table of PCR Primers (based on TrueSeq indices): www.nature.com/nmeth/journal/v13/n11/extref/nmeth.3999-S5.xlsxAd1_noMX:                             AATGATACGGCGACCACCGAGATCTACACTCGTCGGCAGCGTCAGATGTGAd2.1_TAAGGCGA                 CAAGCAGAAGACGGCATACGAGATTCGCCTTAGTCTCGTGGGCTCGGAGATGTAd2.2_CGTACTAG                 CAAGCAGAAGACGGCATACGAGATCTAGTACGGTCTCGTGGGCTCGGAGATGTAd2.3_AGGCAGAA                 CAAGCAGAAGACGGCATACGAGATTTCTGCCTGTCTCGTGGGCTCGGAGATGTAd2.4_TCCTGAGC                 CAAGCAGAAGACGGCATACGAGATGCTCAGGAGTCTCGTGGGCTCGGAGATGTAd2.5_GGACTCCT                 CAAGCAGAAGACGGCATACGAGATAGGAGTCCGTCTCGTGGGCTCGGAGATGTAd2.6_TAGGCATG                 CAAGCAGAAGACGGCATACGAGATCATGCCTAGTCTCGTGGGCTCGGAGATGTAd2.7_CTCTCTAC                 CAAGCAGAAGACGGCATACGAGATGTAGAGAGGTCTCGTGGGCTCGGAGATGTAd2.8_CAGAGAGG                CAAGCAGAAGACGGCATACGAGATCCTCTCTGGTCTCGTGGGCTCGGAGATGTAd2.9_GCTACGCT                 CAAGCAGAAGACGGCATACGAGATAGCGTAGCGTCTCGTGGGCTCGGAGATGTAd2.10_CGAGGCTG             CAAGCAGAAGACGGCATACGAGATCAGCCTCGGTCTCGTGGGCTCGGAGATGTAd2.11_AAGAGGCA             CAAGCAGAAGACGGCATACGAGATTGCCTCTTGTCTCGTGGGCTCGGAGATGTAd2.12_GTAGAGGA             CAAGCAGAAGACGGCATACGAGATTCCTCTACGTCTCGTGGGCTCGGAGATGTAd2.13_GTCGTGAT             CAAGCAGAAGACGGCATACGAGATATCACGACGTCTCGTGGGCTCGGAGATGTAd2.14_ACCACTGT             CAAGCAGAAGACGGCATACGAGATACAGTGGTGTCTCGTGGGCTCGGAGATGTAd2.15_TGGATCTG             CAAGCAGAAGACGGCATACGAGATCAGATCCAGTCTCGTGGGCTCGGAGATGTAd2.16_CCGTTTGT             CAAGCAGAAGACGGCATACGAGATACAAACGGGTCTCGTGGGCTCGGAGATGTAd2.17_TGCTGGGT             CAAGCAGAAGACGGCATACGAGATACCCAGCAGTCTCGTGGGCTCGGAGATGTAd2.18_GAGGGGTT             CAAGCAGAAGACGGCATACGAGATAACCCCTCGTCTCGTGGGCTCGGAGATGTAd2.19_AGGTTGGG             CAAGCAGAAGACGGCATACGAGATCCCAACCTGTCTCGTGGGCTCGGAGATGTAd2.20_GTGTGGTG             CAAGCAGAAGACGGCATACGAGATCACCACACGTCTCGTGGGCTCGGAGATGTAd2.21_TGGGTTTC             CAAGCAGAAGACGGCATACGAGATGAAACCCAGTCTCGTGGGCTCGGAGATGTAd2.22_TGGTCACA             CAAGCAGAAGACGGCATACGAGATTGTGACCAGTCTCGTGGGCTCGGAGATGTAd2.23_TTGACCCT             CAAGCAGAAGACGGCATACGAGATAGGGTCAAGTCTCGTGGGCTCGGAGATGTAd2.24_CCACTCCT             CAAGCAGAAGACGGCATACGAGATAGGAGTGGGTCTCGTGGGCTCGGAGATGT