To begin, take the scarce cell's 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-annealed 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 thermoblock.
Place the tube in a magnet and transfer the supernatant containing the library to a new 1.5 milliliter DNA low binding tube.
