Trick to capturing gene expression and epigenetics from the same cell – it's not rocket science, it's multiomics

Simultaneous Profiling of Gene Expression and Chromatin Accessibility in Single Cells

ATAC-RNA Joint Profiling Workflow

  • Though this could work for any type of gDNA library, e.g. CUT&Tag for profiling histone marks and TF binding)

Sorting and DSP Fixation

  1. Sort 50,000-100,000 cells into RPMI + 10% FBS
  2. Spin down 300g 5 min
  3. Resuspend in 1 ml cold 1X PBS
  4. Spin down 300g 5 min
  5. Gently resuspend in 200uL 1X DSP (https://www.thermofisher.com/order/catalog/product/22585#/22585)
  6. Incubate at RT for 30 min
  7. Quench with 4.1 uL Tris-HCl pH 7.5, mix gently, and keep at 4C
  8. Transfer to BSA-coated 1.5 mL tube tube and top up to 1 mL with FACS buffer (1X PBS, 2.5% FBS, 2.5 mM EDTA)

Transposition

  1. Pellet cells at 500g at 4 C for 5 min in a fixed angle centrifuge
  2. Aspirate all supernatant, carefully avoiding cell pellet, using two pipetting steps (aspirate down to 100 uL with a P1000 pipette and remove final 100 uL with a P200 pipette).
  3. Resuspend cell pellet in 50 uL of transposition mix (1X TD, 0.3X PBS with 0.1% Tween- 20, 0.01% Digitonin and 5 uL TDE) by pipetting up and down 6 times.
  4. Incubate reaction at 55°C for 10 minutes in a thermomixer with 800 RPM mixing
  5. Add 50 uL tagmentation stop buffer (10 mM Tris pH 8, 20 mM EDTA) and incubate on ice for 10 min
  6. Add 150uL FACS buffer and 5 uL 50 ug/mL DAPI before sorting

Capture Beads Preparation

  • Have Streptavidin Magnetic Beads (NEB S1420S)
  1. Wash 400 uL 4 mg/mL streptavidin beads (New England BioLabs) with twice with binding buffer
  2. Resuspend in 368 uL binding buffer + 32 uL 100 uM biotin-oligo-dT
  3. Incubate for 10 min at RT
  4. Wash twice with binding buffer
  5. Resuspend in 460 uL binding buffer + 2.5 mM dNTPs, 1 U/uL RRI

Sorting, Decrosslink, and Amplification

  1. Sort single cells (gate on positive DAPI signal) in a 96 well plate with 5 uL sorting mix (20 mM Tris pH 8, 50 mM DTT, 0.2% SDS, 50 mM NaCl, 2 ug/mL Prot K, 1.2 U/uL RRI)
  2. Spin down plate 1000g for 3 min right after sorting, store at -80C if not immediately processed
  3. Decrosslink at 37 C for 30 min with 800 RPM mixing
  4. Add 4 uL 10% Tween to each well, let stand at RT for 2 min
  5. Add 4 uL of capture beads, incubate 72C for 3 min with 800 RPM mixing
  6. Place on magnet, transfer supernatant to separate plate (ATAC plate)
  7. Wash three times with 4 uL strep bead wash buffer (4 μL 20 mM Tris pH 8, 10 mM DTT, 3.3 mM MgCl2, 33 mM NaCl, 0.5% Tween-20), add to ATAC plate; Resuspend RNA plate (with the beads) in 11 uL RT mix and proceed with the Smartseq2 protocol
  8. Add 2 uL 10 uM mixed primers (indexed P5 and P7)
  9. Add 25 uL 2X KAPA HiFi Mastermix (with 0.05X ROX, 0.1X EVA-Green) to each well then amplify on a qPCR machine
  10. Stop amplification when single cell wells reach threshold
  • Thermal program: 72C for 5:00, 98C for 0:45, 25-30 cycles of (98C for 0:15, 63C for 0:30, 72C for 1:00), 72C for 5:00

ATAC-seq Library Clean-up

  1. Pool PCR products from all wells
  2. Add 5 volumes of buffer PB (Qiagen), and pass through a MinElute column (Qiagen)
    1. Use a vacuum manifold and mount an extension tube
    2. Apply sample to column until everything has passed through
    3. Wash 2x with Buffer PE (Qiagen)
  3. Spin down empty column (16,000g for 1 min) and discard collection tube
  4. Add 17.5 ul Buffer EB (Qiagen) to column and spin down, repeat 3x and combine eluents
  5. Take 50 uL and do double-sided SPRI (0.5X-1.2X), elute in 30 uL Buffer EB
  6. Run library on a hsDNA Bioanalyzer chip (Agilent Technologies)

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