ATAC-Seq Library Preparation of Murine Bone Marrow-Derived Neutrophils

This research endeavors to optimize the ATAC-Seq library construction protocol for investigating neutrophil chromatin accessibility. This optimization will facilitate the study of neutrophil epigenetic reprogramming and reveal their immune response mechanisms under diverse challenges and revealing potential therapeutic targets relevant to the onset and progression of diseases. The average half life of neuro neutrophils is 12.5 hours and their individual half life is even shorter, due to their heightened sensitivity to microenvironmental stimulations.

Consequently, isolating highly active neutrophils and occurring high quality neutrophil nuclei are essential yet challenging steps for the successful construction of ATAC-Seq libraries. We have discovered that immuno magnetic separation enhances the efficiency of neutrophil sorting, ensuring high purity, and providing a scalable protocol for generating high quality ATAC-Seq libraries from rodent neutrophils. To begin, take the euthanized male mouse aged six to eight weeks weighing 19 to 21 grams.

Use small scissors and forceps to carefully separate the femur from the hip joint and tibia. After removing the attached skin and skeletal muscle, rinse the femur with cold PBS in a 10 centimeter Petri dish. Then cut off the femur's epiphyses, and gently insert a 28 gauge needle attached to a two milliliter syringe into the marrow cavity.

Flush out the bone marrow with cold PBS containing 2%fetal bovine serum. Next, use a 70 micrometer cell strainer to filter the cell suspension into a fluorescence activated cell sorting tube. Centrifuge the tube at 500 G for five minutes at 25 degrees Celsius.

After that, carefully remove the supernatant, leaving approximately 100 microliters of liquid at the bottom, and gently tap the tube to loosen the pellet. Then lys the erythrocytes in bone marrow cells using a lysis buffer without polyformaldehyde. Finally, wash the cells twice with RPMI 1640 medium containing 10%fetal bovine serum before resuspending them in a two milliliter volume.

To begin, resuspend the prepared bone marrow cells in two milliliters of RPMI 1640 medium, and centrifuge at 500 G for five minutes at 25 degrees Celsius. Aspirate most of the supernatant, leaving behind approximately 100 microliters and gently tap the tube to loosen the pellet. Next, add 30 microliters of LY-6G beads and gently tap the tube to mix them with the bone marrow cells.

After resuspending the beads in cells in two milliliters of PBS, centrifuge the tube at 500 G for five minutes at 25 degrees Celsius. Remove most of the supernatant and add additional PBS for a final volume of one milliliter. Position the MS column within the magnetic field of a compatible magnetic activated cell sorting separator.

Rinse the column with 500 microliters of PBS and repeat after the complete flow into the MS column. Next, add 500 microliters of the magnetically labeled bone marrow cell suspension to the MS column. Once the column reservoir is empty, refill it immediately.

As soon as the column reservoir is depleted, remove the column from the magnetic field and position it onto a five milliliter tube. After dispensing two milliliters of PBS onto the column, firmly push the plunger into the column to expel the magnetically labeled neutrophils. Centrifuge the 50, 000 single cell suspensions containing neutrophils at 500 G for five minutes at four degrees Celsius in a 0.2 milliliter tube.

Once the supernatant is discarded, resuspend the neutrophils in 50 microliters of pre-cooled lysis buffer. Gently mix the solution using a pipette and incubate on ice for 10 minutes. After centrifuging the neutrophils again and discarding the supernatant, collect the nucleus.

Position the tube on ice before commencing subsequent procedures. To begin, prepare the tagmentation reaction mix containing Tn5 transposase in a PCR tube. Prewarm the refrigerated amplicon purification beads at 25 degrees Celsius for at least 30 minutes.

Resuspend the extracted neutrophil nucleus with 50 microliters of tagmentation reaction mix and gently agitate with a pipette. Centrifuge for five seconds at 2, 600 G.Then incubate the reaction tube in a PCR instrument at 37 degrees Celsius for 30 minutes. Next, add 100 microliters of prevortexed amplicon purification beads to the lysate and mix them thoroughly in the solution.

Incubate the reaction tube at 25 degrees Celsius for five minutes. After centrifuging the tube for five seconds, place the reaction tube on a magnetic rack until the solution becomes clear to purify the interrupted DNA fragment. Carefully remove the supernatant.

Then wash the magnetic beads with 200 microliters of freshly prepared 80%ethanol, while keeping the reaction tube on the magnetic rack for 30 seconds. Afterward, remove the reaction tube from the magnetic rack. Add 26 microliters of double distilled water to cover the magnetic beads, and incubate.

Once the tube is centrifuged, separate the magnetic beads using a magnetic rack until the solution becomes clear. Carefully transfer 24 microliters of supernatant to a new PCR tube. Then add 27.5 microliters of amplicon purification beads to 50 microliters of PCR products and mix them thoroughly in the solution.

Incubate the reaction tube at 25 degrees Celsius for five minutes. Centrifuge the tube for five seconds. Purify the library deoxyribonucleic acid by placing the reaction tube on a magnetic rack until the solution becomes clear.

Carefully transfer the supernatant to a new sterilized PCR tube and discard the magnetic beads. Next, add 50 microliters of amplicon purification beads to the supernatant and mix the magnetic beads thoroughly in the solution. After incubating the reaction tube at 25 degrees Celsius for five minutes, centrifuge for five seconds.

Place the reaction tube on a magnetic rack before removing the supernatant. Wash the magnetic beads twice with 200 microliters of freshly prepared 80%ethanol. Dry the magnetic beads for five minutes with the lid open.

Once the reaction tube is removed from the magnetic rack, add 22 microliters of double distilled water to cover the magnetic beads, and incubate. Separate the beads using a magnetic rack until the solution becomes clear. Carefully transfer 20 microliters of the supernatant to a new sterilized PCR tube before storing it at minus 20 degrees Celsius.

Neutrophil purity was verified to be 98.5%within viable cells after immuno magnetic sorting. ATAC sequencing library DNA showed fragment lengths between 100 and 1, 000 base pairs with no small or large fragment contamination. A typical at ATAC-Seq fragment size distribution with periodic peaks was observed, representing nucleosome free regions, mononucleosomes, dinucleosomes and trinucleosomes.

Compared to the control, LPS stimulated neutrophils showed increased chromatin accessibility in genes such as IL-10, STAT-1, IL12A, CXCL1, and IRAK1.