Strand-Specific Analysis of Proteins at Replicating DNA Strands by Enrichment and Sequencing of Protein-Associated Nascent DNA Method

Our research focus on the mechanism of epigenetic inheritance. Specifically, the DNA replication coupled parental histone recycling process, which represent the first emitting step in epigenetic inheritance. Multiple parental histone deposition which are involved in DNA replication were identified to play important role in parental histone recycling.

This include D helicase complex, DNA polymerase and replication for protection complex. Traditional genetic and biochemical approaches have been values used in this research field. Traditionally, high throughput sequencing methods are being employed to understand the parental histone transfer process.

Before the enrichment and sequencing of protein associated DNA, or experiment method was developed, it was challenging to study the parental histone transfer process. The experiment method was provide powerful tool to address these fundamental questions. To begin, file the yeast cell pellets and re-suspend them by gentle vortexing.

Then wash the same with 0.4 milliliters of CHIP lysis buffer containing protease inhibitors and an antibiotic mix to prevent bacterial contamination. Centrifuge the mixture at 5, 000 G for one minute. Next, add 0.1 milliliters of CHIP lysis buffer with protease inhibitors and antibiotic mix to the pellet.

Followed by approximately 100 microliters of 0.5 millimeter glass beads. Lyse the cells by bead beading for three cycles in a four degree Celsius cold room. Using a 16 gauge hot needle, punch holes in the bottom of the tube.

Collect the lysate by nesting the punctured tube into a 1.5 milliliter empty DNA low binding tube, and centrifuge at 1, 600 G for two minutes at room temperature. Carefully aspirate the supernatant without disturbing the pellet. The cell debris fraction will contain the chromatin.

Re-suspend the cell pellets by pipetting in 0.35 milliliters of supplemented Micrococcal nuclease, or MNase digestion buffer. Next, add 10 units of MNase to the suspension. Mix gently by inverting four to six times, and incubate in a 37 degree Celsius water bath for 20 minutes.

Quench the MNase digestion by adding five microliters of 0.5 molar EDTA to a final concentration of 10 millimolar. Mix gently by inversion, and place the tube on ice for at least 30 minutes. Using 1.5 milliliter Bioruptor microtubes, sonicate the reaction mixture at high power for three minutes with 30 seconds on and 30 seconds off cycles.

Then, centrifuge at 10, 000 G for five minutes at four degrees Celsius. Transfer the supernatant to new tubes and centrifuge again at 10, 000 G for 10 minutes at four degrees Celsius. Then, collect approximately 400 microliters of the supernatant.

Save 30 microliters as input DNA for DNA extraction and freeze at minus 20 degrees Celsius. Use the remaining 370 microliters of cell lysate for histone chromatin immunoprecipitation. To begin, boil about 150 microliters of the previously obtained input and H3K4me3 CHIP samples on a 100 degree Celsius heat block For five minutes, immediately chill the sample on ice for five minutes.

Then, add the required amount of the mentioned components to the sample. Mutate the sample for two hours on a rotor at four degrees Celsius and 10 RPM. Transfer the mixture to a 1.5 milliliter tube containing 20 microliters of washed protein G Sepharose beads.

Mutate the mixture for one hour at four degrees Celsius on the rotor at 10 RPM. Spin the tubes at room temperature at 800 G for one minute. Wash the beads three times with one milliliter of cold Bromo-deoxyuridine, or BrdU immunoprecipitation buffer rotating for three minutes for each wash.

Centrifuge the tube again and wash with one milliliter of TE buffer for three minutes. Spin the tubes again at 800 G for one minute at room temperature, and aspirate the supernatant carefully. Add 100 microliters of TE buffer with 1%SDS to the beads.

Incubate the mixture at 65 degrees Celsius for five minutes, then spin at 800 G for one minute at room temperature. Purify the supernatant into 18 microliters of elution buffer using DNA purification columns to obtain the BrdU immunoprecipitation and H3K4me3 eSPAN samples. Prepare the single-stranded DNA library with the mentioned samples following the ssDNA and low input DNA library preparation kit manual.

Purify the ligation product into 20 microliters of low EDTA Elution buffer. Amplify the DNA library using 50 microliters of reaction mix from the single stranded DNA library preparation kit. Following the displayed cycle, amplify the PCR mix.

Purify the PCR products by mixing them with 60 microliters of beads, prewarmed to 30 degrees Celsius. Mix the PCR products and beads thoroughly. Let the mixture stand at room temperature for five minutes.

Bind the beads on a magnetic stand for three minutes and wash them twice with 200 microliters of freshly prepared 80%ethanol. Air dry the beads for two minutes, and dilute the DNA into 20 microliters of low EDTA buffer provided in the DNA library preparation kit. Measure the library concentration and fragment size distribution using high resolution electrophoresis systems.

Pool equal amounts of individual libraries, including the input H3K4me3 chromatin immunoprecipitation, BrdU immunoprecipitation, eSPAN samples to perform parallel paired end sequencing. A typical gel analysis of the DNA sequencing library of H3K4me3 eSPAN revealed a chromatin nucleosome ladder pattern. The main mononucleosome band appeared at 270 base pairs where the adapters were added to the DNA fragment obtained from digestion.

The different sample mapping revealed peaks at individual nucleosomes surrounding the origin autonomously replicating sequence 607 in wild-type and mcm2-3A cells. The average strand bias revealed that in wild-type yeast cells, parental histone deposition exhibited a slight bias toward the lagging strand. However, in the mutant parental histone H3H4 was transferred to the leading strand.