TITLE: PBMC 10X Genomics Single Cell CUT&Tag Protocol
AUTHORS: Anca B. Mihalas, Hatice S. Kaya-Okur, Steven Henikoff, Anoop P. Patel

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol is based on the bulk CUT&Tag protocol developed in the Henikoff laboratory (</span><a href="https://doi.org/10.1038/s41467-019-09982-5" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">https://doi.org/10.1038/s41467-019-09982-5</span></a><span>) and adapted for single nuclei input onto the 10X Genomics single cell ATAC-seq platform.Throughout this protocol the nuclei will be lost due to multiple washes and partial clumping, so to ensure enough single nuclei are retained, we set up to start with 1 million PBMC-isolated nuclei when starting the primary antibody incubation (B. CUT&Tag protocol, Step 11). To get 1 million PBMC-isolated nuclei aim to input double the amount of live PBMCs, since the nuclei isolation protocol is set up to yield single nuclei solutions by performing low relative centrifugal force (RCF) spins that will prevent nuclei clumping, but will not pellet all nuclei after multiple washes.</span></div></div>

[STEPS]
?. [A. Nuclei isolation protocol]
NOTES:1. All samples and reagents are kept on ice (wet ice) or at 4°C.2. Use low retention tubes (Thermo Scientific # 3451) and pipette tips (Corning # 4140, 4138, 4136), and avoid over pipetting throughout the entire protocol.3. All spins are at 500 RCF for 5 minutes at 4°C, using a swinging-bucket rotor.4. After spins, remove the supernatant carefully, leaving about 50 µl on top of the pellet, and resuspend the pellet in the left-over buffer by tapping the tube.5. If clumps persist, use a wider bore low retention pipet tip (P1000) to gently pipet up and down the nuclei after resuspension in the appropriate buffer. Keep the pipetting of the sample to a minimum that suffices for nuclei pellet dispersion.
?. [A. Nuclei isolation protocol]
In a 1.5 ml tube add 500 μL chilled Nuclei EZ Lysis Buffer to 2 million live PBMCs. Mix the sample by pipetting up and down gently, avoiding bubbles.
?. [A. Nuclei isolation protocol]
Incubate on ice for 5 minutes. Repeat mixing the sample 1-2 times during the incubation.
?. [A. Nuclei isolation protocol]
Filter homogenate using a 70 μm cell strainer (Millipore Sigma # BAH136800070) and transfer to a new 1.5 mL tube.
?. [A. Nuclei isolation protocol]
Centrifuge the nuclei at 500 RCF for 5 minutes at 4°C and remove supernatant leaving about 50 μL above the nuclei pellet.
?. [A. Nuclei isolation protocol]
Add 500 μL Wash Buffer and incubate 5 minutes without resuspending to allow buffer interchange. After incubation, add 1 mL of Wash Buffer and resuspend the nuclei.
?. [A. Nuclei isolation protocol]
Centrifuge the nuclei at 500 RCF for 5 minutes at 4°C, remove supernatant leaving behind about 50 μL and gently resuspend nuclei in 1 mL Wash Buffer.
?. [A. Nuclei isolation protocol]
Repeat STEP 7 and resuspend in 500 μL Wash Buffer. Collect all nuclei by washing off nuclei from the wall of the centrifuge tube.
?. [A. Nuclei isolation protocol]
Filter nuclei with a 40 μm cell strainer (Millipore Sigma # BAH136800040). Visually inspect nuclei integrity under a microscope and count the number of nuclei with a cell counter or hemocytometer.
?. [B. CUT&Tag protocol]
NOTES:1. Use 1 million nuclei per sample.2. Primary antibody: dilute 1:100 in 1ml3. Secondary antibody: dilute 1:100 in 1ml4. pA-Tn5 - MEDS: dilute 1:100 in 1ml5. Use all buffers at room temperature (RT), unless other incubation temperatures are specified.6. Use low retention tubes and pipette tips and avoid over pipetting throughout the entire protocol.7. All spins are at 600 RCF for 3 minutes at RT, unless otherwise specified, using a swinging-bucket rotor. After spins, remove the supernatant carefully, leaving about 50 μl on top of the pellet, and resuspend the pellet in the left-over buffer by tapping the tube.
?. [B. CUT&Tag protocol]
Bind primary antibody:     Prepare 1:100 dilution of primary antibody in 1ml Primary Antibody Incubation Buffer and incubate with 1 million PBMC-isolated nuclei on rotator/nutator at 4°C overnight.
?. [B. CUT&Tag protocol]
Wash:     Spin down nuclei and wash nuclei pellets 2X in 0.5 ml NP40-Dig Wash Buffer.
?. [B. CUT&Tag protocol]
Bind secondary antibody:     Prepare 1:100 dilution of secondary antibody in NP40-Dig Wash Buffer. Add 1ml to each sample. Keep on rotator at RT for 45 minutes.
?. [B. CUT&Tag protocol]
Transition to Medium-salt Wash Buffer from now on. Spin at 600 rcf for 3 minutes. Remove 500 μl supernatant after spin and add 500 μl Medium-salt Wash Buffer (300 mM NaCl).
?. [B. CUT&Tag protocol]
Wash:    Spin down nuclei and wash nuclei pellets 1X with 0.5 ml Medium-salt Wash Buffer.
?. [B. CUT&Tag protocol]
Bind pA-Tn5 - MEDS:     Prepare 1:100 dilution of pA-Tn5 - MEDS complex in Medium-salt Wash Buffer and add 1 ml to each sample. Place on rotator at RT for 1 hour.
?. [B. CUT&Tag protocol]
Wash:    Spin down nuclei and wash nuclei pellets 2X with 0.5 ml in Medium-salt Wash Buffer.
?. [B. CUT&Tag protocol]
Tagmentation:     Add 1 ml Tagmentation Buffer to the sample. Incubate at 37°C for 1 hour. After incubation, add 111 μl 10% BSA/H2O to the 1 ml nuclei in the Tagmentation Buffer (for a final 1% BSA) to prevent nuclei clumping during the following centrifugation step.
?. [B. CUT&Tag protocol]
Transition to 1% BSA/PBS and 4°C from now on. Spin at 600 RCF for 3 minutes at 4°C. Remove 500 μl supernatant after spin and add 500 μl cold 1% BSA/PBS.
?. [B. CUT&Tag protocol]
Wash:    Spin down nuclei at 600 RCF at 4°C and wash nuclei pellets 1X with 0.5 ml in cold 1% BSA/PBS.
?. [B. CUT&Tag protocol]
Count/Strain nuclei:     Resuspend nuclei in a small amount of cold 1% BSA/PBS to aim for a concentration of at least 1100 nuclei/μl if you plan to target 10,000 nuclei recovery. Count the nuclei and inspect for clumps. Filter out clumps through 20-30 μm pore size strainer (Miltenyi # 130-101-812, 130-041-407).
?. [B. CUT&Tag protocol]
Load the nuclei into the 10X Genomics scATAC chip H:https://assets.ctfassets.net/an68im79xiti/7L2MU4QSWfrEgd2h13Efac/d5326fcdc6363aa04e4fdf11b2a1f2f8/CG000209_Chromium_NextGEM_SingleCell_ATAC_ReagentKits_v1.1_UserGuide_RevD.pdf    Follow the 10X Genomics protocol from Step 2, by mixing 15 μl nuclei (e.g. 1020 nuclei/μl for 10,000 targeted nuclei recovery, based on a recovery efficiency factor of 1.53) with 60 μl Barcoding master mix from step 2.1.