Modified ATAC seq protocol 50K HSCs or HPCs

– modified from Corces et al. (Nat Methods 2017)

Sort HSCs/HPCs into 500 µL of PBS + 0.2% BSA

Conventional ATAC-seq prep

  • Spin down 50,000 sorted HSCs or HPCs @ 500g in swinging bucket centrifuge / 10 minutes / 4° C
  • Pipet off supernatant in two steps – first with P1000 and then with P200 to get remaining 10-20 µL
  • Discard supernatant & gently re-suspend in 25 µL of ice cold ATAC-RSB buffer with 0.1% IGEPAL CA-630
    • RSB buffer: 10 mM Tris-Hcl, pH 7.4, 10 mM NaCl, 3 mM MgCl2
  • Spin down at 500g for 15 min at 4° C
  • Carefully discard the supernatant as before
  • With pellet on ice:
    • Prep the reaction mix:
      • 12.5 µL 2x TD/Transposase buffer (from nextera kit)
      • 2.0 µL transposase (from nextera kit)
      • 10.5 µL nuclease-free water
    • Prepare each reaction separately rather than a single master mix, and transfer to pelleted nuclei
  • Gently pipette to re-suspend nuclei in the transposon reaction mix
  • Incubate for 1 hour at 37° C
  • Place immediately on ice
  • Purify using Qiagen MinElute Reaction kit
    • Elute in 11 µL EB (10 mM Tris @ pH 8)
    • Store reaction at -20° C until library prep

OMNI-ATAC prep for small numbers of cells

Typically we use for 10,000 or less, though we obtain similar results with 50,000 cells as with the conventional prep.

  • Spin down HSCs or HPCs @ 500g in fixed angle microfuge/ 5 minutes / 4° C
  • Pipet off supernatant in two steps – first with P1000 and then with P200 to get remaining 10-20 µL
  • Discard supernatant & gently re-suspend in 50 µL of ATAC-RSB buffer with 0.1% NP40, 0.1% Tween-20 and 0.01% digitonin
    • RSB buffer: 10 mM Tris-Hcl, pH 7.4, 10 mM NaCl, 3 mM MgCl2
  • Incubate on ice for 3 minutes, then add 950 µL of RSB with 0.1% Tween-20 nut no NP40 or digitonin, invert 3x and mix
  • Spin down at 500g for 10 min at 4° C in fixed angle centrifuge
  • Carefully discard the supernatant as before
  • Prep the reaction mix:
    Prepare each reaction separately rather than a single master mix, and transfer to pelleted nuclei.
    • 12.5 uL 2x TD/Transposase buffer (from nextera kit)
    • 16.5 µL 1xPBS
    • 0.5 µL 1% digitonin
    • 0.5 µL 10% Tween-20
    • 2.5 µL transposase (from nextera kit)
    • 5 µL nuclease-free water
  • Gently pipette to resuspend nuclei in the transposon reaction mix
  • Incubate for 30 minutes at 37° C
  • Place immediately on ice
  • Purify using Qiagen MinElute Reaction kit
    • Elute in 11 µL EB (10 mM Tris @ pH 8)
    • Store reaction at -20° C until library prep
Library amplification and purification

Amplify library with NebNext

  • 10 µL eluted DNA
  • 10 µL nuclease free water
  • 2.5 µL Ad1
  • 2.5 µL Ad2 (barcode primer)
  • 25 µL Neb Next
    • Perform qPCR with ATAC-seq primers to calculate optimal cycle number.
    • Cycling conditions are: 72°C/5min, 98°C/30sec, (98°C/10sec, 63°C/30sec, 72°C/1min)xN cycles, hold at 4° C

See Buenrostro et al. for ATAC primer sequences and qPCR methods: http://www.nature.com/nmeth/journal/v10/n12/full/nmeth.2688.htm

Perform SPRI cleanup
  • Add 1.8x volume (90 µL) beads per reaction and pipet up and down
  • Incubate at room temp 10 minutes
  • Put on magnet 5 minutes and discard supernatant
  • Wash twice with 200 µL 80% EtOH and discard ethanol
  • Air dry beads 5 minutes
  • Add 50 µL water to the beads and mix
  • Incubate 10 minutes and then return the tubes to the magnet
  • Remove supernatant and save
Size select
  • Add 0.7x volume (35 µL) of beads to the cleaned sample
  • Incubate 10 minutes
  • Put on magnet 5 minutes and transfer the supernatant to a fresh tube
  • Add additional 1.1x of original volume (55 µL) of SPRI beads (1.8x final)
  • Incubate 10 minutes.
  • Put on magnet 5 minutes and discard supernatant
  • Wash twice with 200 µL 80% EtOH and discard ethanol
  • Air dry beads 5 minutes
  • Add 40 µL TE to the beads and mix
  • Incubate 10 minutes and then return the tubes to the magnet
  • Remove supernatant and save

Send samples to GTAC for Agilent analysis and sequencing.