Break-seq and ssDNA-seq

This protocol contains the Break-seq procedures as published in Hoffman et al 2015 Genome Research, as well as the unpublished ssDNA-seq method.
 
Part I.  Sample collection and preparation of yeast DNA embedded in  agarose plugs 

The preparation of agarose plug embedded DNA is adapted from Iadonato, S. P., and A. Gnirke. 1996.  RARE-cleavage analysis of YACs. Methods Mol Biol 54: 75-85.



1.
Yeast cultures are grown in synthetic complete media at 25 oC or 30oC.  Log phase cells (at OD660 of 0.25) are synchronized in G1 by the addition of alpha factor at 200 nM or 3 mM (for bar1 and BAR1 strains respectively), followed by incubation for approximately one and a half generations, or until the unbudded cell population reaches at least 90%.  HU is added at 200 mM (or other replication inhibitors can be used), and cells are released from the G1 arrest by the addition of pronase at 0.02 mg/ml or 0.3 mg/ml (for bar1 and BAR1 strains respectively).
2.
Harvest the cells by adding them to test tubes containing NaN3 on ice such that the final concentration of NaN3 will be 0.1%.  I typically collect 50 ml of each time point sample to make 3 plugs of ~100 ml each.  Scale up for the 0 time point control sample if multiple S phase samples are collected.
3.
Spin down cells at 3000 x rpm in the floor centrifuge for 5 min. Wash the cell pellet twice with 50 mM EDTA, spinning 5 min at 3000 x rpm as above.
4.
Resuspend the cells in 50 mM EDTA to a final concentration of 2 x 109 cells/ml and warm the cell suspension at 45°C for 5 min. Add an equal volume of 1% low-melt InCert (or SeaPlaque) agarose in 50 mM EDTA, also prewarmed to 45°C . (This procedure will make 0.5% plugs, which are fairly fragile, but are better if further manipulations like in gelo digest are required. If not, use 2% agarose to make 1% plugs, which are less fragile). Mix the suspension thoroughly by vortexing and pipette 100 µl aliquots into each plug mold to harden.  Plugs may be allowed to set at room temperature or placed at 4°C (for 15 minutes).
5.
Extrude each plug from the plug mold into a six-well dish, up to 3 plugs per well. To each well add 6 ml of freshly prepared spheroplasting solution. Incubate at 37°C for 4 h with gentle shaking.
6.
Aspirate off the spheroplast solution from each well and add 6 ml of LDS solution. Incubate with gentle shaking at 37°C for at least 15 min. Remove and add fresh LDS solution. Incubate with gentle shaking at 37°C overnight.
7.
Wash the plugs 2 x 30 min with 6 ml of 0.2x NDS solution with gentle shaking at room temperature.
8.
Wash the plugs 3 x 30 min with 6 ml of TE, pH 8.0 with gentle shaking at room temperature. Plugs may be stored at 4°C in six-well dishes with TE, pH 8.0, covered with Saran wrap to prevent excessive evaporation. High-molecular-mass DNA will usually remain undegraded for at least six months.
9.
Refer to the CHEF gel apparatus manual for suggested parameters. To visualize all the yeast chromosomes, we use a switch time ramped from 60-120s, 200V, 24 hours. To obtain better separation of the smaller chromosomes, we use a switch time ramped from 35-70s, 200V, 22 hrs.


Spheroplasting Solution
40 ml 1M Sorbitol (approx. 1M final conc.)
1.6 ml 0.5M EDTA, pH 8.0 (20mM final)
0.4 ml 1M Tris-HCl, pH7.5 (10mM final)
40 µl 2-mercaptoethanol (14mM final)
0.5 mg/ml Zymolyase 20-T

NDS Solution
0.5 M EDTA
10 mM Tris base
1% Sarkosyl
(pH 9.5)
 
To 350 ml H2O add 93 g Na2EDTA•2H2O and 0.6g Tris base
Adjust the pH to greater than 8.0 with 100 to 200 pellets of solid NaOH
Add 50 ml of 10% N-lauryl sarcosine
Adjust the pH to 9.5 with concentrated NaOH
Bring the final volume to 500 ml with H2O
Filter-sterilize and store at room temperature.

LDS Solution
1% lithium dodecyl sulfate
100 mM EDTA
10 mM Tris-HCl, pH 8.0
Per 1 liter:
10 g lithium dodecyl sulfate (Sigma Chemical Cat #L-4632)
200 ml 0.5M EDTA, pH8.0
10 ml 1M Tris-HCl, pH8.0
Bring volume to 1 liter with H2O, filter-sterilize, and store at room temperature

 


Part II.  In-gel random-primed labeling of ssDNA or End-repair labeling of DSBs with Biotin-dNTPs for Illumina Sequencing


1.
In-gel labeling of ssDNA by Klenow:
1) Make 1X reaction buffer from 10X stock reaction buffer (5 ml per agarose plug of 100 ml volume):
50 mM   Tris-HCl pH 6.8
5 mM     MgCl2
10 mM   β-mercaptoethanol
*no hexamers in this buffer
2) Remove agarose plugs to new dishes (6-well plates, one plug per well)
3) Add 5 ml TE 0.1 to each well and incubate on rotor at RT for 15 min
4) Aspirate buffer.  Repeat c.
5) Add 5 ml 1X reaction buffer and incubate on rotor at RT for 30 min
6) Aspirate buffer.  Repeat e.
7) During the last wash step, prepare labeling reaction mix:
Estimate gel volume approx. 50 ml
Mix (50 ml) contains:
5 μl     Biotin-dNTPs (1 mM each)
20 μl   2.5X reaction buffer (from the ArrayBox)
3 μl     Klenow (high concentration, 50,000U/ml)
22 μl   H2O
8) Assemble a small water bath.  Use a 1ml Blue tip box, remove insert, place a CLEAN small gel tray inside, add approx. 50 ml of agd H2O or enough to submerge the gel tray legs halfway.  Cut a piece of parafilm and use gloved hands to place it on the gel tray.  You could mark the positions/sample names on the edges of the parafilm to indicate where the plugs go.
9) Using a CLEAN spatula move the plugs onto the parafilm according to the arranged positions.
10) Pipet 50 μl of reaction mix onto each agarose plug slowly. 
*I try to let the liquid hover on top of the agarose but you may get some overflow on the side.  However, surface tension will hold the liquid near the plugs. 
11)  Incubate at 37oC for 2-3 hours.
1a.
In-gel labeling of DSBs by End-Repair Enzyme mix:
1) Make 1X reaction buffer from 10X stock reaction buffer:
33 mM Tris-Acetate pH 7.8
66 mM KAc
10 mM Magnesium acetate
0.5 mM DTT
2) Transfer agarose plugs to new dishes (6-well plates, one plug per well)
3) Wash each plug 2X with 5 ml TE 0.1 on rotator at RT for 15 min each.
4) Wash each plug 2X with 5 ml 1X reaction buffer on rotator at RT for 30 min each.
5) During the last wash step, prepare labeling reaction mix:
Estimate gel volume approx. 50 μl
Mix (50 ml) contains:
  5 μl   End-repair buffer (10X)
  5 μl   dNTP mix containing Biotin-14-dATP (1 mM each)
  5 μl   10 mM ATP
  3 μl   End-It enzyme mix
32 μl   H2O
6) Set up the labeling reaction by adding 50 μl of labeling mix to each plug in humidity chamber as described above.
7) Incubate at RT or in the 23°C incubator (if RT is low) for 1.5 hrs.
8) Wash 2X with 5 ml TE 0.1 for 15 min each.
9) Equilibrate 2X with 200 μl 1X β-Agarase buffer on ice for 30 min each.
2.
β-Agarase digestion:
1) Transfer plugs to epi tubes
2) Melt at 70°C for 10 min in water bath, mix occasionally by flicking
3) Transfer to 42°C for 5 min
4) Add 3 μl β-Agarase to each plug and incubate at 42°C for 1 hr.
5) Add 100 μl β-Agarase buffer (total volume approximately 200 ml)

3.
Sonicate (using Covaris M220) and Clean-up DNA:
1) Transfer 100 μl of the β-Agarase-digested DNA sample from step 2 into a Covaris snap-cap tube (microtube AFA Fiber Pre-slit Snap-Cap 6x16 mm, Covaris #520045)
2) Sonicate using the preloaded protocol named “DNA_300_bp_130_μl_Snap_Cap_microtube”
3) Transfer sonicated DNA to a new epi tube
4) Transfer the remaining ~100 μl β-Agarase digested DNA from step 2 to the same snap-cap tube above
5) Repeat sonication using the same preloaded protocol above
6) Pool the sonicated DNA with like sample into the epi tube above
7) Repeat for all samples
8) Run 5 μl on 1.2% agarose gel to confirm the breakage.  DNA should be sheared to an average size of < 500 bp (typically we get 400 bp average size)
9) Use Qiagen PCR cleanup kit to clean the samples (follow instructions).  If the DNA quantity is much greater than 20 μg then multiple columns would be required. 
*The maximum load we have tried is 15 μg per column without significant loss.
10) Elute each sample with 50 μl of EB.  Nanospec DNA concentration to analyze potential loss rate during purification.  DNA can be stored at 4°C until the next step.

4.

End-Repair

Sheared DNA (8-11 mg of DNA) and H2O               67 μl
10X End repair Buffer                                                   10 μl
2.5 mM dNTP mix                                                           10 μl
10 mM ATP                                                                       10 μl
End it enzymes                                                                   3 μl
1. Mix well and incubate at RT or in the 23°C incubator if RT is too low for 45 minutes.
2. Purify through a Qiagen PCR purification column.  Elute DNA in 32 μl EB.

5.

A-tailing:  Either with Taq DNA polymerase or Klenow fragment of Pol I

A-tailing with Taq

DNA (end-repaired)                                                             32 μl
10X PCR buffer w/Mg2+ (Roche 12779800)                10 μl
50 mM MgCl2                                                                            3 μl
100 mM dATP (Invitrogen P/N55082)                         0.5 μl
Taq (NEB M0267S, 5 units/ml)                                       0.5 μl
H2O                                                                                           56 μl
1.     Mix well and incubate at 70oC in the PCR machine for 30 minutes.
2.     Wash 2X with 200 μl 1X B&W
3.     Wash 2X with 200 μl EB
4.     Resuspend in 10 μl EB

 

A-tailing with Klenow

DNA (end-repaired)                                                             32 μl
10X reaction buffer (NEB E6055A, see below)              5 μl
Klenow  (NEB E6054A, 5 units/ml)                                  3 μl
H2O                                                                                           10 μl
1.     Mix well and incubate at 37oC for 30 minutes. 
2.     Purify through a Qiagen PCR purification column.  Elute DNA in 52 μl EB.
3.     Nanospec (using up to 2 μl) the DNA sample for total input DNA onto M270 Dynabeads.

6.

Streptavidin beads purification

1.   Add 50 ml EB to each DNA sample to make up final volume of 100 μl
2.   Use 50 μl of M270 Dynabeads suspension per sample.  Mix the Dynabeads suspension well by swirling the bottle.  Remove 50 μl of beads from vial and place on magnet for 2 min and then remove supernatant.
*The binding capacity of the beads for dsDNA is ~10 mg/mg beads.  Assuming 5 μg of DNA in each agarose plug, one needs 0.5 mg beads.  The concentration of M270 is 10 mg/ml.  Thus, 50 μl of beads is enough for 5 μg of biotinylated DNA.  This amount far exceeds the usual level of biotin labels in our sample (10-30% depending on the treatment) with total 10 μg of DNA.
3.   Add 100 μl 1X B&W buffer, mix with pipette 12 times
4.   Place on magnet for 2 min and then remove supernatant
5.   Repeat steps 3 and 4 twice
6.   Add 100 μl (twice the original volume, final concentration 5 mg/ml) 2X B&W buffer, mix with pipette 12 times
7.   Add equal volume (100 μl) of DNA, mix with pipette 12 times
8.   Incubate at RT on roller for 30 mins
9.   Place on magnet for 2 min. 
10. Transfer flow through to a new epi tube and Nanospec to estimate the amount of immobilized DNA (~10%).
11. Wash 2X with 100 μl 1X B&W buffer
12. Wash 2X with 100 μl EB
13. Resuspend beads in ligation mix (see below).

2X B&W buffer (200 ml)
10 mM Tris-HCl, pH 7.5                                2 ml 1M stock
1 mM EDTA, pH8.0                                       1 ml 200 mM stock
2 M NaCl                                                        80 ml 5M NaCl

Add agd H2O from tank to 200 ml final volume, filter sterilize, store at RT



7.


Ligation of adaptors 

DNA (end-repaired and A-tailed)                                                              
50 mM Solexa-1                                                           3 μl
50 mM Solexa-2                                                           3 μl
10X T4 ligast buffer                                                    5 μl
T4 DNA ligase                                                               3 μl
H2O                                                                               37 μl
Mix well and incubate at RT over night.
Wash 2X with 200 μl 1X B&W
Wash 2X with 200 μl EB
Resuspend in 50 μl of EB
 
8.

PCR enrichment on beads

*Use < 200ng of DNA (on the M270 beads) for a 50-μl PCR reaction.  Could use either Cloned Pfu or the KAPA HiFi HotStart ReadyMix as enzyme.
DNA on the beads                       10 μl              DNA on the beads                             10 μl
10X Pfu buffer                                5 μl              2X HotStart Ready Mix                   25 μl
10X dNTPs (2.5mM each)          5 μl               Solexa-F (10 μM)                             1.5 μl
Solexa-F (10 μM)                    1.25 μl              Solexa-R (10 μM)                             1.5 μl
Solexa-R (10 μM)                   1.25 μl                H2O                                                       12 μl
Pfu                                                  0.5 μl
H2O                                                 27 μl

Reaction conditions:
98oC    1 min                                                           98°C     5min
20 cycles of:                                                           18 cycles of:
98oC   30 sec                                                           98°C    20 sec
65oC   30 sec                                                           65°C    15 sec
72oC   2 min                                                            72°C     1 min
72oC   5 min                                                            72°C     5 min
4oC ∞                                                                        4oC      

Run 2 μl on a 1.5% agarose gel.  Pipette the rest of the PCR product without the beads into a fresh epi tube.
9.

Purification using AMPure beads (to remove free adaptors)

1.   Swirl the AMPure bottle to resuspend the magnetic particles

2.   Use 1.8 volumes of AMPure beads per 1 volume of DNA solution (90 μl for each 50 ml PCR reaction).  Transfer beads to PCR product in an epi tube.
3.   Mix the AMPure beads and PCR product thoroughly by pipette mixing 10 times and vortexing for 10 sec.
4.   Incubate at RT for 10 min, with frequent mixing by vortexing.
*This step is important to ensure sufficient binding of DNA to the beads.  DO NOT put the mix on magnet right away.
5.   Place the epi tube on magnet for 5 min.
6.   Aspirate the sup and transfer to a fresh epi tube to save as flow through for later analysis.
7.   Without removing the tube from the magnet, add 200 µl of 70% ethanol onto the beads and incubate at RT for 30 sec.  Aspirate sup and discard.
8.   Repeat 7.
9.   Leave the cap open to air-dry for 20 min.
10. Elute DNA by adding 40 μl of agd H2O or TE and pipette mix 10 times and vortex for 10 sec.
11. Incubate at RT for 10 min, with frequent mixing by vortexing.
*The sample can be stored at 4°C overnight.
12. Place on magnet for 5 min.
13. Transfer sup to new epi tube.
14. Run 1 μl of the eluted DNA as well as the flow through on 1% agarose gel to check.
10.

Making Solexa adaptors

1.   Prepare all four primers (Solexa_1_top, 1_bottom, PlexP2_top and PlexP2_bottom) at 100 μM in TE pH 8.0 and store at 4oC

2.   Mix equal volumes of Solexa_1-top and Solexa_1-bottom (50 μl each)

3.   Mix equal volumes of Solexa_PlexP2_top and Solexa_PlexP2_bottom (50 μl each)

4.   Incubate at 95oC in PCR machine for 5 min

5.   Turn off machine at the end of the 5 min and let it cool to RT gradually (about 1.5 hours) and store at –20oC



Solutions needed:

2.5x reaction buffer for Klenow
            125 mM         Tris-HCl pH 6.8
            12.5 mM         MgCl2
            25 mM            β-mercaptoethanol
            750 mg/ml     random hexamers

10X reaction buffer for Klenow labeling
            500 mM         Tris-HCl pH 6.8
            50 mM            MgCl2
            100 mM         β-mercaptoethanol
            Mix the following:
            1 M Tris-HCl pH 6.8                            25 ml
            100 mM MgCl2                                    25 ml
            12.5 M β-mercaptoethanol              400 μl
           
10X reaction buffer for End-repair labeling
            330 mM         Tris-Acetate pH 7.8
            660 mM         KAc
            100 mM         MgAc2
            5 mM              DTT
            Mix the following:
            1 M Tris-Acetate pH 7.8                   16.5 ml
            Potassium acetate solid                    3.24 g
            Magnesium acetate-4H2O                1.07 g
            1 M DTT                                                250 μl
            agd H2O to final vol. of 50 ml
                                               
Biotinylated dNTP mix (1 mM each)
            1 mM each     dTTP, dCTP, dGTP
            0.84 mM         dATP
            0.16 mM         Biotin-14-dATP (Invitrogen Cat. 19524-016)
            Mix the following:
            0.4 mM Biotin-14-dATP      125 μl
            100 mM dATP                       2.44 μl
            100 mM dGTP                       2.94 μl
            100 mM dCTP                       2.94 μl
            100 mM dTTP                       2.94 μl
            H2O                                     157.74 μl
            Total                                          294 μl

10X NEB Next dA-Tailing reaction buffer:
            100 mM Tris-HCl pH 8
            100 mM MgCl2
            500 mM NaCl
            10 mM DTT
            2 mM dATP
            Mix the following:
            1 M Tris-HCl pH 8                 100 μl
            1 M MgCl2                               100 μl
            5 M NaCl                                  100 μl
            1 M DTT                                     10 μl
            100 mM dATP                          20 μl
            H2O                                           670 μl

Random hexamers (IDT)
MW = 1,791.7
1 mM stock = 1.79 mg/ml

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