Detection of nucleosome positions

Nucleosome Preparation Protocol for S. cerevisiae

Xu Zhou 07/10/2020

xu.zhou@yale.edu; xuzhou1984@gmail.com

CELL PREPARATION

1)    Grow cells (1 L culture) in 10 mM phosphate media to an OD600 = 0.4

2)    Take 500 mL of culture, filter and transfer to pre-warmed 800 mL SD no phosphate media and SD 10 mM phosphate media. (Starve for 60 minutes)

3)    After starvation, for 800 mL of culture, add 22 mL of 37% w/v formaldehyde.  Swirl intermittently for 15 min at RT.

4)    After 15 minutes, add 43 mL of 2.5 M glycine to quench formaldehyde.  Swirl 5 minutes at RT.

5)    Spin cells at 6000 rpm for 5 minutes at 4 degrees Celsius.

6)    Pour off supernatant and bring pellet up in 10 mL of 10 mM Tris pH 7.5, 100 mM NaCl.  Transfer to falcon tube and bring to final volume of 30 mL.

7)    Spin at 2800 rpm for 5 minutes at 4 degrees Celsius.

8)    Pour off supernatant and add another 30 mL of 10 mM Tris, 100 mM NaCl.

9)    Spin again at 2800 rpm for 5 minutes.

10)   Pour off liquid and dry pellet at RT for 10 minutes.

11)   Freeze tubes and put at -80 degrees Celsius overnight.

CHROMATIN EXTRACTION

Buffer A                                                   Stock                                       Volume

50mM Hepes pH 7.5                                1M Hepes pH 7.5                    500 uL

150mM NaCl                                            1M NaCl                                 1.5 uL

2 mM EDTA                                             500mM EDTA                        40 L

1% triton X-100                                        Commercial Stock                  100 uL

.1% Sodium Deoxycholate                        Commercial Stock                  10 mg

Water                                                                                                         7.85 uL

Complete protease inhibitors                    One tablet                                __________

                                                                                                                  10 mL

Mnase reaction buffer                            Stock                                       Volume

10mM Tris pH 7.5                                    1M Tris pH 7.5                       100 uL

50mM NaCl                                              1M NaCl                                 500 uL

5mM MgCl₂                                              1M MgCl₂                               50 uL

1mM CaCl₂                                               1M CaCl₂                                10 uL

1 mM b-ME                                              14.2M b-ME                           .7 uL

.1% Igepal                                                 Commercial Stock                  10 uL              

Water                                                                                                         9.33 mL

                                                                                                                  10 mL

buffers are made fresh before use.

1)    Remove frozen tubes and thaw at RT in RT water.  Add 200 mL of buffer A to each tube.  Transfer to a 2 mL screw cap tube (~750 mL of buffered cells).  Add acid washed glass beads to the top of the tube, leaving just a small space at the top.  Put tubes into bead beater at 4 degrees Celsisus on maximum speed.  Run 5 cycles of 1 minutes on and 2 minutes off for the old beadbeater.

2)    Poke a hole in the bottom of the tubes with a 20 G1 needle and place them into 1.5 mL tubes.  Then place conjoined tubes into a 50 mL falcon tube.  Spin tubes at 2000 rpm for 5 minutes at 4 degrees Celsius.

3)    Remove 1.5 mL tube with lysed cell extract.  Add 500 mL MNase reaction buffer.  Pipette gently to mix.  Spin at 16,000xg for 2 minutes at RT.

4)    Remove supernatant and add a new 500 mL MNase buffer.  Pipette gently to mix.  Spin at 16,000xg for 3 minutes at RT.  Remove supernatant.

5)    Add a new 1 mL of MNase buffer (pellet has shrunk upon mixing) or bring the total volume to 1.5 ml.  Mix gently by pipetting.  Transfer 750 mL to 2 separate tubes.

6)    Make two MNase digestion reaction with high and low MNase concentration: Add 0.5 uL of MNase (100 U/l ) (low) to one tube and 1 uL (high) to the other.  Put tubes at 37 degrees Celsius for 25 minutes.  Afterwards add 15 uL of 500 mM EDTA, vortex and put on ice for 10 minutes.  Spin at 16,000xg for 8 minutes at 4 degrees Celsius.  Transfer supernatant to a new 1.5 mL tube (discard pellet).The volume is about 650 mL. Add 16 mL of 20% SDS ( 0.5% final) and vortex. Add 30 mL of Proteinase K and put tubes at 65 degrees Celsius overnight to reverse cross-links.

NUCLEOSOMAL PURIFICATION:

1)    Remove samples from 65 degrees Celsius and spin at 16,000xg for 3 minutes.  Transfer supernatant to a phase lock gel heavy tube that has been pre-spun at 16,000xg for 1 min.  Add 50 uL of 5M NaCl and 700 mL 25:24:1 phenol/chloroform/isoamyl alcohol and mix by inversion.

2)    Spin tubes at 16,000xg for 3 minutes and transfer supernatant to a new phase lock gel heavy tube. (Pre-spun at 16,100xg for 1 minute).  Add 700 mL of phenol/chloroform and mix by inversion.  Spin again at 16,100xg for 3 minutes.

3)    Transfer supernatant (~600 mL) to a 2 mL eppendorf tube with 1.2 mL 100% ethanol and put at -20 degrees Celsius for 30 minutes.  Spin at 16,100xg for 8 minutes at 4 degree.  Afterwards, remove ethanol and add 100 mL of 0.2 mg/mL RNase A in TE buffer and put tubes at 37 degrees Celsius for 1 hour. The pellet is hard to dissolve. Incubation in 37 degree water bath helps to get it dissolved.

4)    Use Qiagen PCR purification kit to purify samples using standard protocol (attached). Use at least 2 tubes per sample. Load the flowthrough from the first tube into a new spin column. Elute each in 50 mL buffer EB. (The capacity of PCR purification column is 10 mg. Assuming 300 Da per nt, yeast genome is 13Mb, and DNA mass per Mol cell is roughly 300*13*2*10⁶ = 10¹⁰ Da.  1 OD unit is roughly 10⁷ cells. For 200 OD unit cells, the total DNA mass is 200*10⁷*10¹⁰/(6.02*10²³) = 3*10^-5 g = 30 mg. Considering inefficient cell lsysis, MNase digestion, 2 tubes of DNA purification column would be safe to recycle all nucleosomal DNA)

5)    Add 10 mL of 6X loading dye (green one) and load samples onto a 1.5% TAE-agarose gel.  Load the sample on big gel using the thin comb. Run at 160V for 35 minutes and stain in ethidium bromide for 30 minutes.  Took a fast picture with limited UV and excised the mononucleosomal band (corresponding to 150 base pair size).

6)    Took excised monoculeosomal DNA bands, chop up into smaller fragments, and load into a Quantum Pre^® DNA freeze N’ Squeeze tube (Bio-Red, 732-6165). Put tubes at -20 degrees Celsius for 1 hour, spin at 16,000 g for 7 min. Remove column with remaining agarose, add 1 ml 100% EtOH, 50 ul 2M Ammonium Acetate pH 4.6, mix well by inversion and put at  -20 degrees Celsius for 30 min. Spin at 16,000 g for 10 min at 4 degrees Celsius. Wash DNA pellet with 1ml 70% EtOH and spin again at 16,000 g for 5 min. Remove all liquid and air dry the pellet.

7)    Use 15 ul buffer EB to dissolve DNA pellet and combine two tubes together for each sample. Remove 3.5 ul nucleosomal DNA sample and combine with 2.5 ul 6x loading dye, load all 5 ul onto a 1.5% TAE agarose gel. Run the gel at 130 U for 25 min and stain in 1x EtBr for 30 min, and take a picture to visualize the nucleosomal DNA band.

8)    Save the DNA for illumina library preparation.

LIBRARY PREPARATION AND SEQUENCING:

For each experiment, 0.5 μg of mono-nucleosomal DNA (~5 pmol) was used for sequencing library construction according to the Illumina Truseq paired-end protocol. ~1 pmol of paired-end ligated library was recovered from the agarose gel for each experiment before PCR amplification. Since the amount of ligated library was sufficient for sequencing, we only performed 4 cycles of PCR amplification with the Illumina primer cocktail to obtain a double stranded sequencing library. The number of sequencing clusters generated on the flow-cell represented at most 1/10,000 of the amplified library, such that the chance was very small to have more than one copy of duplicated library molecules clustered on the flow-cell (p<1.8 × 10^–5, binomial distribution). Therefore, we treated each paired-end read as an individual nucleosome from a single cell. Libraries were sequenced with an Illumina Hiseq 2000 and the paired-end sequencing reads were aligned to the S. cerevisiae genome Scer02 with bowtie 2.0. We mapped reads with two or fewer mismatches, and insert lengths were restricted to sizes between 100 bp and 300 bp. Reads with multiple reportable alignments were randomly assigned to one such alignment. The midpoints of all sequenced DNA fragments were randomly rounded to an integer genomic coordinate and used to generate sequencing read midpoint occupancy maps.

BAYESIAN DECONVOLUTION FOR NUCLEOSOME POSITIONS

Please refer to the codes and methods for Bayesian deconvolution of nucleosome positioning at https://github.com/airoldilab/cplate, developed by Alex Blocker and Edoardo M Airoldi (current email address: airoldi@temple.edu). The readme file contains walk through of applying the deconvolution methods for identifying nucleosome positions.

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