With LeTech, the main question that we are trying to answer is how the chromatin interacts in the nucleus and therefore, to understand a new layer of gene regulation. We have proven with LeTech that we can explore the genome organization of a given sample, link the non-coding mutation associated with disease with potential genes affected, and detect chromosome rearrangements such as translocation, for example, in tumor samples. Now we can explore the 3D chromatin organization of scar cell types, such as primary stem cells or relevant clinical samples.
Our protocol reduces the input materials and the time and cost needed to obtain quality data to explore the genome organization at the restriction fragment resolution. LeTech is expanding the reach of the 3D chromatin organization field, allowing us to explore new cell types previously impossible to analyze. Thanks to LeTech, the scientific community can explore the special regulation of gene expression, for example, in malignant transformation in a specific chromosome population inside a tumor.
To begin, take an eloquat of frozen scarce cells and add one milliliter of cold lysis buffer to disrupt the cell membrane. Incubate the cells on ice for 30 minutes, mixing by inversion every five minutes. Centrifuge the nuclei for 10 minutes at 1000 G, remove the supra natant and re-suspend the nuclei in 500 microliters of cold 1.25 x restriction buffer 2.
Then, add 5.5 microliters of 10%sodium dodecyl sulfate. Mix by vortexing and incubate in a thermo block. Then quench the SDS by adding 37.5 microliters of 10%tritton X 100.
Mix and incubate the sample as demonstrated earlier. Add 7.5 microliters of hint three to digest the chromatin, mix the solution and incubate overnight at 37 degrees Celsius. To begin, take the digested scarce cells DNA and place it on ice.
Prepare a master mix by using the reagents required to fill in and biotinylate the restriction fragment overhangs. Incubate the sample at 37 degrees Celsius for 75 minutes, mixing by inversion every 15 minutes. Chill the sample on ice and ligate the filled in DNA ends by preparing a master mix using the reagents required for ligation.
Finally, incubate the sample for four to six hours at 16 degrees Celsius, and then for 30 minutes at room temperature. Add 30 microliters of 10 milligrams per milliliter of proteinase K to de cross-link the chromatin. Mix the solution and incubate overnight at 65 degrees Celsius.
After purifying the DNA, begin by taking ligated and de cross-linked digested chromatin of the scarce cells. Set the water bath sonicater at a duty factor of 20%Peak incidents power of 50 to 200 cycles per burst for 65 seconds, and a temperature range of 6 to 10 degrees Celsius, and sonicate the ligated and de cross-linked digested chromatin. After end repairing the sample, transfer 150 microliters of C1 streptavidin beads per sample to a 1.5 milliliter tube.
Place them on a 1.5 milliliter tube magnet and wait until all the beads are stuck to the wall. Then remove the supernatant, leaving the beads behind. Next, wash the beads by adding 400 microliters of tween buffer and resuspending them with soft vortexing.
Place the tube back in the magnet until all the beads are stuck to the wall. Then remove the supernatant, leaving the beads behind. After resuspending the beads in 300 microliters of 2x no tween buffer or NTB, combine them with the 300 microliters of the sample.
Finally, incubate for 15 minutes, rotating at room temperature to pull down the informative DNA fragments with biotin. To begin, take the scarce cells DNA fragments pulled with biotin. Prepare a master mix by adding the reagents for dATP-tailing and incubate the sample for 30 minutes at 37 degrees Celsius.
Then inactivate klenow exo-minus by incubating the sample for 10 minutes at 65 degrees Celsius and cooling it on ice. After washing the beads with 300 microliters each of TB, NTB, and 100 microliters of ligation buffer, resuspend them in 50 microliters of ligation buffer. Add four microliters of 15 micromolar pre-akneeled adapter mix and one microliter of 2000 units per microliter T4 DNA ligase to the sample.
Wash the beads with 400 microliters of TB, 200 microliters of NTB, and 100 microliters of restriction buffer two. After amplifying the library by PCR, pool all the 50 microliter reactions from the same sample into a 1.5 milliliter DNA low binding tube. Place it on a tube magnet and wait until all the beads are stuck to the wall.
Transfer the supernatant containing the library to a new 1.5 milliliter DNA low binding tube and top up with TLE buffer to 500 microliters. To perform a double-sided selection using paramagnetic bead purification, add 200 microliters of stock beads to the library, mix by vortexing, and incubate for 10 minutes, rotating at room temperature. Then place the library on a magnet and wait until all the beads are stuck to the wall.
Transfer the supernatant containing the library to a new 1.5 milliliter DNA low binding tube. Concentrate the beads by taking 750 microliters of stock beads and placing them in a 1.5 milliliter tube on a magnet. Once all the beads are stuck to the wall, remove the supernatant and resuspend the beads by vortexing in 300 microliters of new stock beads.
Add 300 microliters of concentrated beads to the sample. Mix by vortexing and incubate for 10 minutes, rotating at room temperature. Place on a magnet.
Wait until all the beads are stuck to the wall and remove the supernatant. Wash the beads by adding one milliliter of 70%ethanol to the tube with the beads still on the magnet. Allow the beads to air dry and resuspend them in 21 microliters of TLE buffer by vortexing.
To elute the library from the beads, incubate the sample for 10 minutes at 37 degrees Celsius in a thermo block. Place the tube in a magnet and transfer the supernatant containing the library to a new 1.5 milliliter DNA low binding tube. Begin by taking 500 to 1000 nanograms of the scarce cell DNA library.
Dry the DNA with a vacuum concentrator and resuspended in 3.4 microliters of nuclease free water. After adding the blockers and while on the thermocycler at 65 degrees Celsius, transfer the hybridization solution with the biotinylated RNA to the blocked library. After closing the tube lid firmly, incubate in the thermocycler overnight at 65 degrees Celsius.
Then transfer 50 microliters of T1 streptavidin beads per sample to a 1.5 milliliter DNA low binding tube, and place them on a 1.5 milliliter tube magnet. Once all the beads are stuck to the wall, remove the supernatant, leaving the beads behind. Now wash the beads thrice with 200 microliters of the binding buffer from the target enrichment kit and resuspend the beads in 200 microliters of binding buffer.
Transfer the sample to the resuspended beads while on the thermocycler and incubate for 30 minutes. After washing the beads with 200 microliters of wash buffer one, incubate them for 15 minutes, rotating at room temperature. Then wash the beads thrice with 200 microliters of wash buffer two heated to 65 degrees Celsius and incubate in a thermo block.
Finally, after amplifying the library by PCR, perform a DNA purification using paramagnetic beads by adding 270 microliters of stock beads to the sample and mixing by vortexing. An automated electrophoresis profile from a library just before capture provided 49.7 nanograms per microliter of DNA in 20 microliters reaction volume. However, 3.06 nanograms per microliter of DNA is obtained from a high sensitivity automated electrophoresis profile from a finished lech library.
