1. Thaw sperm aliquot(s) containing at least 250,000 sperm cells (typically 10 uL) at room temperature.
2. Quick spin sperm aliquot and transfer to nonstick 1.5 mL tube.
3. Spin for 10 minutes at 400g at 4 degrees Celsius. Remove supernatant.
4. Resuspend sperm pellet in 10 uL PBS.
5. Repeat spin from (3). Resuspend sperm pellet in 2.5 uL Buffer SMT.
6. Submerge tube for 5-10 seconds in liquid nitrogen. Uncap tube to de-pressurize. Hold tube in fist until thawed.
7. Repeat (6) for a total of 3 freeze-thaw cycles.
8. Make 100 uL Buffer DB with freshly added heparin and BME (enough for 4 samples): combine 1.5 uL 10 mM heparin, 2 uL 100 mM BME, and 96.5 uL Buffer DB.
9. Add 22.5 uL of Buffer DB with heparin and BME from (8) to each sperm sample.
10. Incubate at 37 degrees Celsius for 45 minutes. Thaw heparinase on ice during the latter half of this incubation.
11. Remove sample(s) to room temperature. Add 5 uL freshly-thawed heparinase and pipette gently to mix.
12. Incubate sample with added heparinase at room temperature for 2 hours before preceding immediately to next steps.
13. Generate a DNA-stained 1:100 dilution of sperm (for counting under fluorescence microscope) by combining 1 uL sperm mixture, 10 uL 10X SYBR I, and 89 uL PBS. Allow to incubate in the dark at room temperature, with or without gentle rotation, for 5-10 minutes.
14. Add 20 uL diluted, stained sperm to Fuchs-Rosenthal C-chip hemocytometer and count the number of sperm present in 5 big squares under green fluorescent light. (Sperm appear as bright fluorescent dots; un-decondensed sperm are compact dots while sperm at this state, after decondensation, are bigger circles.) Sperm concentration per microliter is the number of sperm in 5 big squares multiplied by 100. (Or, use fluorescence to count sperm via a known and trusted method.)
15. If using 10X Genomics GemCode (old) reagents (for other reagents continue to step 16):
a. Dilute sperm to a final number of 10,833 in 5 uL ultrapure water.
b. Make master mix: per sample, combine 32.5 uL GemCode reagent mix, 1.5 uL primer release agent, 9.2 uL GemCode polymerase, and 16.8 uL ultrapure water.
c. Add 60 uL of master mix to each sperm sample, pipetting gently with wide bore pipettes to mix.
d. Vortex GemCode beads at full spead for 25 seconds.
e. Dilute vortexed GemCode beads 1:11 ultrapure water (to a total volume of at least 90 uL per sample).
f. Prepare chip for droplet generation (per 10X Genomics’ protocol): add 60 uL of sample-master mix mixture in the sample wells, then 85 uL of freshly-pipette-mixed 1:11 diluted-bead mixture to the bead wells, then 150 uL droplet generation oil to the oil wells of the chip.
g. Generate and process droplets following 10X GemCode User Guide Rev C Steps 5.36-5.5.2.
h. Complete library generation following 10X GemCode User Guide Rev C Steps 5.6-6.2.6.
16. If using 10X Genomics Chromium Single-cell DNA (new) reagents (if using previous reagents follow step 15 instead): Follow 10X Chromium Single Cell DNA User Guide Rev B “Chromium Single Cell DNA Reagents kits” protocol for generating single-cell sequencing libraries (Step 1- “Cell Bead Generation,” Step 2 “Cell Bead Processing,” Step 3 “GEM Generation & Barcoding,” Step 4 “Post GEM Incubation Cleanup & QC”, and Step 5 “Library Construction”), using the prepared sperm as input. Target 2000 cells for recovery. This process can be paused at any point recommended by 10X.
17. Sequence according to 10X’s and Illumina sequencer instruction. (Generally, sequence yield is improved by increasing the input library concentration slightly from that recommended by Illumina.)
18. Process sequence data as described in Bell et al “Insights about variation in meiosis from 31,228 human sperm genomes” (Nature 2020).