TITLE: sciMAP-ATAC
AUTHORS: Andrew Adey, Casey Thornton

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">High-throughput single cell genomic assays resolve the heterogeneity of cell states in complex tissues, however, the spatial orientation within the network of interconnected cells is lost. As cell localization is a necessary dimension in understanding complex tissues and disease states, we present a tool for highly scalable spatially-resolved single cell profiling of chromatin state. We use high density multiregional sampling to perform single-cell combinatorial indexing on Microbiopsies Assigned to Positions for the Assay for Transposase Accessible Chromatin (sciMAP-ATAC) to produce single-cell data of equivalent quality to non-spatial single-cell ATAC-seq.</div></div>

[STEPS]
?. [Prepare Nuclei Isolation Buffer]
Construct 50mL Nuclei Isolation Buffer (NIB):  ABC1Final
ConcentrationStock
ConcentrationVolume of Stock210 mM Tris HCl, pH 7.51M
Tris-HCl, pH7.5500 uL310 mM NaCl5M
NaCl100 uL43mM MgCl21M
MgCl2150 uL50.1 % Igepal10%
Igepal500 uL60.1 % Tween10%
Tween500 uL7ddH20to 50mL (add 48.25mL)Store NIB on ice throughout nuclei dissociation and preparation of tagmentation plates.
ABC1Final
ConcentrationStock
ConcentrationVolume of Stock210 mM Tris HCl, pH 7.51M
Tris-HCl, pH7.5500 uL310 mM NaCl5M
NaCl100 uL43mM MgCl21M
MgCl2150 uL50.1 % Igepal10%
Igepal500 uL60.1 % Tween10%
Tween500 uL7ddH20to 50mL (add 48.25mL)
OPTIONAL: To prevent protease degradation, we also add 2 tablets of Pierce Preotease Inhibitor Tablets, EDTA-Free to NIB following construction. Vortex to fully dissolve tablets.
NIB is stable at    for at least 1 month without noticeable degradation in library quality or nuclei dissociation ability.
?. [Isolate nuclei]
Nuclei isolation from cryopreserved histological sectionsIf sample is sourced from microbiopsy of a cryopreserved histological section, dissociate cells using NIB incubation and trituration (described below).This protocol is optimized for brain tissue microbiopsies. Additional optimization may need to be performed for other tissues.1. Prepare 96-well plate(s) for microbiopsy punchesPipette 100 uL NIB into each well. Number of wells corresponds to number of punches to be collected.Seal plate and store on ice until ready to collect microbiopsies2. Prepare instruments & tissue for collecting microbiopsies.Transfer cryopreserved tissue sections from -80C freezer on to dry ice in an insulated containerLoad Palkovitz punch handle with selected diameter punch (options: 250 um, 500 um, 750 um, 1 mm, 1.25 mm)Prechill Palkovitz punch by placing the punch in dry ice3. Collect microbiopsies in a cryostat at -20CPlace tissue cryosection slide in cryostat and allow ~1 min to acclimateLocate region of interest and collect punchDeposit punch in well of 96-well plate by depressing punch plunger. (Ensure that punch enters well)Repeat for each region to be resected. Place each new punch in new wellReseal 96-well plate(s) 4. Dissociate and wash microbiopsiesShake plate on ice for 1 hour at 80 rpmUsing a multi-channel pipettor, triturate each well 30x.  Spin down plate for 10 min at 500 rcf at 4CUsing a multi-channel pipettor, aspirate 90 uL of supernatant. 5. Dilute microbiopsy nuclei to desired concentration Prepare 70 uM Pitstop 2 + NIB master mix: For one plate, combine 168 uL 3 mM Pitstop2 & 3,432 uL NIBAdd 30 uL of 70 uM Pitstop 2 NIB master mix to each well and triturate to resuspend cells6. Split punches into multiple platesSplit 40 uL of resuspend cells into 4 new 96-well (DNA/protein Lo-bind) plates with 10uL diluted cells/well.2. Prepare instruments & tissue for collecting microbiopsies.Transfer cryopreserved tissue sections from -80C freezer on to dry ice in an insulated containerLoad Palkovitz punch handle with selected diameter punch (options: 250 um, 500 um, 750 um, 1 mm, 1.25 mm)Prechill Palkovitz punch by placing the punch in dry ice3. Collect microbiopsies in a cryostat at -20CPlace tissue cryosection slide in cryostat and allow ~1 min to acclimateLocate region of interest and collect punchDeposit punch in well of 96-well plate by depressing punch plunger. (Ensure that punch enters well)Repeat for each region to be resected. Place each new punch in new wellReseal 96-well plate(s) 4. Dissociate and wash microbiopsiesShake plate on ice for 1 hour at 80 rpmUsing a multi-channel pipettor, triturate each well 30x.  Spin down plate for 10 min at 500 rcf at 4CUsing a multi-channel pipettor, aspirate 90 uL of supernatant. 5. Dilute microbiopsy nuclei to desired concentration Prepare 70 uM Pitstop 2 + NIB master mix: For one plate, combine 168 uL 3 mM Pitstop2 & 3,432 uL NIBAdd 30 uL of 70 uM Pitstop 2 NIB master mix to each well and triturate to resuspend cells6. Split punches into multiple platesSplit 40 uL of resuspend cells into 4 new 96-well (DNA/protein Lo-bind) plates with 10uL diluted cells/well.3. Collect microbiopsies in a cryostat at -20CPlace tissue cryosection slide in cryostat and allow ~1 min to acclimateLocate region of interest and collect punchDeposit punch in well of 96-well plate by depressing punch plunger. (Ensure that punch enters well)Repeat for each region to be resected. Place each new punch in new wellReseal 96-well plate(s) 4. Dissociate and wash microbiopsiesShake plate on ice for 1 hour at 80 rpmUsing a multi-channel pipettor, triturate each well 30x.  Spin down plate for 10 min at 500 rcf at 4CUsing a multi-channel pipettor, aspirate 90 uL of supernatant. 5. Dilute microbiopsy nuclei to desired concentration Prepare 70 uM Pitstop 2 + NIB master mix: For one plate, combine 168 uL 3 mM Pitstop2 & 3,432 uL NIBAdd 30 uL of 70 uM Pitstop 2 NIB master mix to each well and triturate to resuspend cells6. Split punches into multiple platesSplit 40 uL of resuspend cells into 4 new 96-well (DNA/protein Lo-bind) plates with 10uL diluted cells/well.4. Dissociate and wash microbiopsiesShake plate on ice for 1 hour at 80 rpmUsing a multi-channel pipettor, triturate each well 30x.  Spin down plate for 10 min at 500 rcf at 4CUsing a multi-channel pipettor, aspirate 90 uL of supernatant. 5. Dilute microbiopsy nuclei to desired concentration Prepare 70 uM Pitstop 2 + NIB master mix: For one plate, combine 168 uL 3 mM Pitstop2 & 3,432 uL NIBAdd 30 uL of 70 uM Pitstop 2 NIB master mix to each well and triturate to resuspend cells6. Split punches into multiple platesSplit 40 uL of resuspend cells into 4 new 96-well (DNA/protein Lo-bind) plates with 10uL diluted cells/well.Spin down plate for 10 min at 500 rcf at 4CUsing a multi-channel pipettor, aspirate 90 uL of supernatant. 5. Dilute microbiopsy nuclei to desired concentration Prepare 70 uM Pitstop 2 + NIB master mix: For one plate, combine 168 uL 3 mM Pitstop2 & 3,432 uL NIBAdd 30 uL of 70 uM Pitstop 2 NIB master mix to each well and triturate to resuspend cells6. Split punches into multiple platesSplit 40 uL of resuspend cells into 4 new 96-well (DNA/protein Lo-bind) plates with 10uL diluted cells/well.5. Dilute microbiopsy nuclei to desired concentrationPrepare 70 uM Pitstop 2 + NIB master mix: For one plate, combine 168 uL 3 mM Pitstop2 & 3,432 uL NIBAdd 30 uL of 70 uM Pitstop 2 NIB master mix to each well and triturate to resuspend cells6. Split punches into multiple platesSplit 40 uL of resuspend cells into 4 new 96-well (DNA/protein Lo-bind) plates with 10uL diluted cells/well.6. Split punches into multiple plates
NoteIsolation of nuclei is dependent on the sample being used. And optimization should be performed. Below we list two example nuclei isolation protocols to act as general use for cell culture and primary tissue samples. Tissue should follow a dounce homogenization protocol, while liquid cell cultures can be pelleted and resuspended directly in NIB.
Note: Keep a record of slide number, punch location, and well ID for each punch.
Note: Pipette gently in order to reduce bubbles and to prevent nuclei shearing
Note: Pellet will not be visible. Be careful to not touch sides of bottom while drawing off supernatant.
Note: We find that for microbiopsy punches from 200 um thick tissue /250 um biopsy punch results in (thousand nuclei):Min: 6, 1st Q: 12, Median: 15, Mean: 16.85, 3rd Q: 22.25, Max: 29We want 10 μL nuclei well. Each punch dissociation can be split into 4 wells (4.2K nuclei/reaction). Therefore, we want 40 μL of 4,200 nuclei/10 μL:C1V1 = C2V2(1,685 nuclei/μL)(10μL) = (421.25 nuclei/μL)(x μL)x = 40 μLVolume to add: 40 μL - 10 μL (residual volume) = 30 μLFinal concentration of Pitstop 2 should be 70 μM in 40 μL of resuspended nuclei. Therefore:C1V1 = C2V2(3000 μM)(x μL) = (70 μM)(30 μL); x = 1.43 μL Therefore, for each well, add: (1.4 μL 3 mM Pitstop 2 + 28.6 μL NIB) = 30 μL of 70 μM Pitstop 2 NIBThis should be done by making a master-mix. Given 1 plate (96 wells), prepare a master-mix for 120 wells:(168 μL 3mM Pitstop 2 + 3,432 μL NIB) = 3600 μL of 70 μM Pitstop 2 NIB, for one plate.
Note: Make sure to keep well ID consistent between plates.
?. [96-plex Tagmentation]
Prepare tagmentation plateAdd the following reagents to diluted nuclei in 96-well plate(s) (DNA and Protein Lo-bind):         Prepare 70 μM Pitstop 2  + 2X TD buffer Master Mix for one plate:  28 μL 3mM Pitstop2 + 1,200 μL 2X TD Add  to each well Add  to each wellSpin down plate for  at 500 rcf at
[70uM Pitstop 2/TD buffer (2X)]
[8uM uniquely indexed transposase]
[min]
4 °C
Tagmentation can be performed by the addition of 1 µL of 2.5 µM barcoded transposome (EZ-Tn5 variant) (Amini et al. 2014)
?. [96-plex Tagmentation]
TagmentationSeal plate and incubate at  for Place plate on ice immediately to stop reaction.
[with gentle shaking (300 rpm on themomixer)]
?. [96-plex Tagmentation]
Pool all wells for second sortPool all wells into 15mL conical tube, while maintaining everything on ice.Add 2uL/per mL pooled sample of DAPI (5mg/mL) and bring to sorter for second sort.
?. [96-plex PCR]
Preparing Second Plate of Transposase Neutralization Buffer (8.5uL/well):  ABC1Final ConcentrationStock ConcentrationVolume of Stock20.59 mg/mL20 mg/mL BSA0.25 uL30.059% (w/v)1% SDS (w/v)0.5 uL4to 8.5 uLddH2O7.75 uLAdd 2.5 uL of 10 uM i5 Indexed PCR Primer and 2.5 uL of 10 uM i7 Indexed PCR Primer prior to sort.
ABC1Final ConcentrationStock ConcentrationVolume of Stock20.59 mg/mL20 mg/mL BSA0.25 uL30.059% (w/v)1% SDS (w/v)0.5 uL4to 8.5 uLddH2O7.75 uL
?. [2nd Sort Protocol]
Flow sort single nuclei based on DAPI gateUsing the same gates as first sort, sort X nuclei per well into prepared second plate with modified sort settings:"Single cell" rather than "Normal"            This leads to a higher abort count (less efficient sorting) but is more precise in quantificationKeep sorted samples on ice to prevent transposases cross-reacting with other nuclei.            This leads to a higher abort count (less efficient sorting) but is more precise in quantificationExample gating strategy using the Sony SH800 Flow sorter:Spin down plate at 500 rcf for  at  to ensure nuclei are properly suspended in solution.
Sort X nuclei per well (X is dependent on number of wells tagmented in first sort, as a linear trend)•	96 wells (1 plate)  = 22 nuclei/well for PCR•	144 wells (1 and 1/2 plates) = 33 nuclei/well for PCR•	192 wells (2 plates) = 44 nuclei/well for PCR etc...
[min]
4 °C
Volume added, even by sorting 100 nuclei is minimal in our hands and does not require concentration adjustments.
?. [Transposase Denaturation]
Transposase DenaturationDenature remaining transposase in sorted nuclei using SDS mixture on Eppendorf Thermocyclers. for
55 °C
[min]
?. [96-plex PCR]
Amplifying single cell libraries
Nextera PCR Mater Mix currently produces the highest quality libraries. An alternative master mix using Kapa Hifi Non-Hotstart has been developed and produces good results.
?. [96-plex PCR]
Add 13.5 uL PCR Master Mix to each wellPerform Real-time PCR on the Bio-Rad CFX Connect:Pull once majority of well begin to plateau. Sci-ATAC libraries amplify between 14-22 cycles dependent on nuclei per well. Store libraries at   or
[NPM]
[100X SYBR Green I]
[dH2O]
[for 6 months]
[forever]
?. [Library Clean-up and Quantification]
Pool post-PCR ProductPool 10 uL from each well into 15mL conical tube.
?. [Library Clean-up and Quantification]
Concentrate DNA via column clean upRun full pool volume through Qiaquick PCR purification column following manufacturer's protocol.Elute in
[10 mM Tris-HCl pH 8.0]
?. [Library Clean-up and Quantification]
Clean by size selection with SPRI beadsPerform a 1X SPRI bead size selection (selecting for DNA > 200 bp).Add   to column elution, once beads are at room temperature.Let mixture incubate at room temperature for Place tube on magnetic rack and wait for magnetic beads to pellet and elution to fully clear (roughly )Remove full volume of elution without disrupting bead pellet.Resuspend bead pellet in freshly prepared Remove full volume of elution without disrupting bead pellet.Let beads fully air dry (roughly  )Resuspend beads off magnetic rack in Let mixture incubate at room temperature for   for DNA to fully become suspended.Place tube on magnetic rack and wait for magnetic beads to pellet and elution to fully clear (roughly )Remove full volume of elution without disrupting bead pellet and move to clean tube.
[18% PEG SPRI Beads]
[min]
[min]
[80% ethanol (v/v)]
[min]
[10 mM Tris-HCl pH 8.0]
[min]
[min]
?. [Library Clean-up and Quantification]
Qubit DNA HS QuantificationQuantify DNA concentration with 1uL eluted sample on Qubit DNA High-sensitivity kit following manufacturer's protocol.
?. [Library Clean-up and Quantification]
Agilent DNA HS Bioanalyzer QuantificationDilute sample to 4 ng/uL based on read out of Qubit by addition of 10mM Tris-HCl pH 8.0.Run 1 uL sample on Agilent DNA HS Bioanalyzer following manufacturer's protocol.
?. [Sequencing]
Custom Nextseq500 Chemistry ProtocolSequence libraries on a NextSeqTM 500 sequencer (Illumina Inc.) running NextSeq500 NCS (v4.0) software loaded within a range of 1.2-1.6 pM with a custom sequencing chemistry protocol (Read 1: 50 imaged cycles; Index Read 1: 8 imaged cycles, 27 dark cycles, 10 imaged cycles; Index Read 2: 8 imaged cycles, 21 dark cycles, 10 imaged cycles; Read 2: 50 imaged cycles) using custom sequencing primers.Oligonucleotide IDOligonucleotide SetSequence 5'>3'Read1_sequencing_PrimerRead1_IlluminaNextSeq500_SequencingPrimerGCGATCGAGGACGGCAGATGTGTATAAGAGACAGRead2_sequencing_PrimerRead2_IlluminaNextSeq500_SequencingPrimerCACCGTCTCCGCCTCAGATGTGTATAAGAGACAGIndex1_sequencing_PrimerIndex1_IlluminaNextSeq500_SequencingPrimerCTGTCTCTTATACACATCTGAGGCGGAGACGGTGIndex2_sequencing_PrimerIndex2_IlluminaNextSeq500_SequencingPrimerCTGTCTCTTATACACATCTGCCGTCCTCGATCGC
Oligonucleotide IDOligonucleotide SetSequence 5'>3'Read1_sequencing_PrimerRead1_IlluminaNextSeq500_SequencingPrimerGCGATCGAGGACGGCAGATGTGTATAAGAGACAGRead2_sequencing_PrimerRead2_IlluminaNextSeq500_SequencingPrimerCACCGTCTCCGCCTCAGATGTGTATAAGAGACAGIndex1_sequencing_PrimerIndex1_IlluminaNextSeq500_SequencingPrimerCTGTCTCTTATACACATCTGAGGCGGAGACGGTGIndex2_sequencing_PrimerIndex2_IlluminaNextSeq500_SequencingPrimerCTGTCTCTTATACACATCTGCCGTCCTCGATCGC
?. [96-plex PCR]
Add 13.5 uL PCR Master Mix to each wellPerform Real-time PCR on the Bio-Rad CFX Connect:Pull once majority of well begin to plateau. Sci-ATAC libraries amplify between 14-22 cycles dependent on nuclei per well. Store libraries at   or
[5X Kapa Hifi Buffer (GC Buffer)]
[10 mM Kapa dNTP Mix]
[1 U/uL Kapa Hifi DNA Polymerase]
[100X SYBR Green I]
[dH2O]
[for 6 months]
[forever]
Non-hotstart polymerase is critical for this. As persisting gaps in the genomic DNA have to be filled prior to amplification. This is done by the polymerase at lower temperature (72C for 5 min) prior to the actual PCR.
?. [Library Clean-up and Quantification]
Pool post-PCR ProductPool 10 uL from each well into 15mL conical tube.
?. [Library Clean-up and Quantification]
Concentrate DNA via column clean upRun full pool volume through Qiaquick PCR purification column following manufacturer's protocol.Elute in
[10 mM Tris-HCl pH 8.0]
?. [Library Clean-up and Quantification]
Clean by size selection with SPRI beadsPerform a 1X SPRI bead size selection (selecting for DNA > 200 bp).Add   to column elution, once beads are at room temperature.Let mixture incubate at room temperature for Place tube on magnetic rack and wait for magnetic beads to pellet and elution to fully clear (roughly )Remove full volume of elution without disrupting bead pellet.Resuspend bead pellet in freshly prepared Remove full volume of elution without disrupting bead pellet.Let beads fully air dry (roughly  )Resuspend beads off magnetic rack in Let mixture incubate at room temperature for   for DNA to fully become suspended.Place tube on magnetic rack and wait for magnetic beads to pellet and elution to fully clear (roughly )Remove full volume of elution without disrupting bead pellet and move to clean tube.
[18% PEG SPRI Beads]
[min]
[min]
[80% ethanol (v/v)]
[min]
[10 mM Tris-HCl pH 8.0]
[min]
[min]
?. [Library Clean-up and Quantification]
Qubit DNA HS QuantificationQuantify DNA concentration with 1uL eluted sample on Qubit DNA High-sensitivity kit following manufacturer's protocol.
?. [Library Clean-up and Quantification]
Agilent DNA HS Bioanalyzer QuantificationDilute sample to 4 ng/uL based on read out of Qubit by addition of 10mM Tris-HCl pH 8.0.Run 1 uL sample on Agilent DNA HS Bioanalyzer following manufacturer's protocol.
?. [Sequencing]
Custom Nextseq500 Chemistry ProtocolCustom primers and sequencing protocol for sci-atac libraries.To be expanded.
?. [Loading Tn5 Enzyme with sci-protocol Oligonucleotides]
Prepare ReagentsPrepare   for protein dilution.   ABCD1ReagentStock ConcentrationFinal ConcentrationAmount of Stock2HEPES-KOH (pH 7.2)1M100mM5mL3NaCl5M200mM2mL4Glycerol100%25%12.5mL5Triton-X100100%0.2%100uL6ddH2O30.4mL (to 50mL)7DTTDry2mM15.4 mg
[2.125X Tn5 Dilution buffer]
ABCD1ReagentStock ConcentrationFinal ConcentrationAmount of Stock2HEPES-KOH (pH 7.2)1M100mM5mL3NaCl5M200mM2mL4Glycerol100%25%12.5mL5Triton-X100100%0.2%100uL6ddH2O30.4mL (to 50mL)7DTTDry2mM15.4 mg
?. [Loading Tn5 Enzyme with sci-protocol Oligonucleotides]
Prepare Mosaic End reverse compliment (ME'), i7,  i5 oligonucleotides at See attached spreadsheet for oligonucleotide sequences. Three sets of oligonucleotides are listed for both i5 and i7 Tn5 loading. This yields (3 i5 sets) x (3 i7 sets)=9 uniquely identifiable 96 well plates or 864 unique well barcode combinations.Mosaic End oligonucleotide sequence used for Tn5 loading is also listed within the spreadsheet.
[Tris-HCl buffer (pH 8.0)]
Synthesis quality of these oligonucleotides is critical. HPLC purification is essential. We find that Eurofins oligos outperform IDT by roughly 10 fold in library complexity.
All indexes are designed to be 2 or greater Hamming distance from all others to allow for sequencing errors.
?. [Loading Tn5 Enzyme with sci-protocol Oligonucleotides]
Anneal Indexed Oligoes to Mosaic End Reverse ComplimentPreparation of dsDNA through annealing. Volumes are adjusted for a single 96-well plate loading.  AB112.5 uL100 uM i5 Tn5 Indexed oligo212.5 uL100 uM Mosaic End Reverse Compliment oligo353.125 uL2.125x Tn5 Dilution Buffer2. For each i7 barcoded oligo prepare the following reaction (12 total):  AB18.5 uL100 uM i7 Tn5 Indexed oligo28.5 uL100 uM Mosaic End Reverse Compliment oligo336.125 uL2.125x Tn5 Dilution Buffer
AB112.5 uL100 uM i5 Tn5 Indexed oligo212.5 uL100 uM Mosaic End Reverse Compliment oligo353.125 uL2.125x Tn5 Dilution Buffer
AB18.5 uL100 uM i7 Tn5 Indexed oligo28.5 uL100 uM Mosaic End Reverse Compliment oligo336.125 uL2.125x Tn5 Dilution Buffer
?. [Loading Tn5 Enzyme with sci-protocol Oligonucleotides]
Anneal Oligo mixtures within a Thermocycler with the following reaction.Slow ramp down to This results in annealed oligo species per reaction (i7/ME' and i5/ME').
95 °C
[at a rate of -2.5C/min]
[hold]
Oligoes should be freshly annealed prior to loading Tn5 transposome
?. [Loading Tn5 Enzyme with sci-protocol Oligonucleotides]
Plate Annealed OligosPrepare a 96-well plate with the following loading schema.1. Add  of i5/ME' () to each respective wells in a row-wise fashion.2. Add  of i7/ME' () to each respective wells in a column-wise fasion.This results in  at /well
5 µl
5 µl
[i5/ME' and i7/ME' Indexed Oligos]
?. [Loading Tn5 Enzyme with sci-protocol Oligonucleotides]
Adjust Salt Concentration on Tn5 and LoadPrepare Tn5 protein as described in "Generation and Purification of pTXB1.Tn5" protocol.Prior to loading Tn5 protein adjust NaCl concentration. Combine:  AB11152 uL1152 uL2144 uL144 uL
AB11152 uL1152 uL2144 uL144 uL
?. [Loading Tn5 Enzyme with sci-protocol Oligonucleotides]
Add  of salt-corrected Tn5 to each well of the 96 well plate.Assemble the Tn5/oligo mixture via incubation at  for  .
12 µl
25 °C
Store at -20C for no more than 8 months.