TITLE: SHARE-seq protocol v2.2
AUTHORS: Amelia Hall

[DESCRIPTION]
TBD

[STEPS]
SECTION: 1.1 Ordering Oligo Plates and oligos for plate production
2. One of the more involved aspects of SHARE-seq (and SPLiT-seq) is properly making the oligo hybridization 96 well plates - this section covers that in detail before moving into any of the day to day aspects of this protocol (those start at step 24, in section 2).
 
In summary, this section will walk you through ordering deepwell 96 well oligo plates for all 3 rounds of hybridization in SHARE-seq (or SPLiT-seq), with an additional set of 96 oligos that can be ordered ("plate 2"), plus linker oligos.

In the original SPLiT-seq paper the authors calculated a barcode collision rate above ~5% with greater than around 25,000 cells or nuclei in a cellular sub pool (these are generated at the end of the first day of SHARE-seq).  So for experiments using a singular set of three rounds of 96 barcodes, don't create cellular sub pools greater than 25,000 cells - the rate of barcode collisions will be high.  If a second plate of 96 barcodes is used (we refer to these as "plate2"), and the two plates are pooled together and intermixed after each round of hybridization, we determined that up to 100,000 cells or nuclei can be pooled together safely, with low (<5%) risk of barcode collision.
SECTION: 1.2 Annealing Oligo plates - making oligo plates
5. This part occurs only after you have all of the oligo deepwell plates and the Round 1,2,3 linker oligos from IDT.  If you don't have these yet, don't proceed with platemaking!  

The recipe below is for eighteen 96 well plates for each round of barcoding (54 total plates per set of 96 oligos, that's 108 if you're making both plate1 and plate2).  We recommend that you use different color PCR plates for each round of each plate. Conveniently, ThermoFisher offers 96 well skirted plates in 6 different colors, search for part number AB2396N (where N=R is red, G=green, Y=yellow, B=blue, O=orange - the base model is clear).  Please be sure you purchase part number AB2396, and not AB3396 - those plates have white wells that will make it hard to tell how much fluid is in the well.
SECTION: 1.1 Ordering Oligo Plates and oligos for plate production
3. Ordering oligo deepwell plates from IDT:

Using the attached Excel file, batch order the oligos on the tabs labeled "Plate1 R1 96", "Plate1 R2 96" "Plate1 R3 96" from IDT in a 96 well "deepwell" plate.   If 192 barcodes are desired (2 plates), then order the oligos on the tabs labeled "Plate2 R1 96", "Plate2 R2 96", "Plate2 R3 96." Order at 25nM yield, 100uM concentration, resuspended in IDTE buffer, in a 96 well plate format.  An example of what the plate upload looks like is shown below.  Note that the R3 plate(s) will be less expensive than the R1 or R2 plates, since there is no 5' phosphate base needed in these molecules.

  

 
 
 
SECTION: 1.1 Ordering Oligo Plates and oligos for plate production
4. You'll also need linkers to anneal with the oligos in each plate to create the unique cellular barcode during the hybridization of SHARE.  Order the oligos below from IDT, order the linker oligos at 1mM (1000uM) concentration, and the blocking oligos at 100uM; order oligos resuspended in IDTE. 

Note: the 1uM is the "scale" of what you're ordering from IDT, not the concentration!  The concentration is "formulation" from IDT, and that's 100uM in IDTE for the blocking oligos, and 1000uM in IDTE for the linker oligos.
                  Name   LENGTH   Sequence   Scale   Purification   Specific oligos   order from IDT      Round 1 linker   30   CCGAGCCCACGAGACTCGGACGATCATGGG   1um   STD      Round 1 linker,CCGAGCCCACGAGACTCGGACGATCATGGG,1um,STD      Round 2 linker   30   CAAGTATGCAGCGCGCTCAAGCACGTGGAT   1um   STD      Round 2 linker,CAAGTATGCAGCGCGCTCAAGCACGTGGAT,1um,STD      Round 3 linker   30   AGTCGTACGCCGATGCGAAACATCGGCCAC   1um   STD      Round 3 linker,AGTCGTACGCCGATGCGAAACATCGGCCAC,1um,STD      Round 1 blocking   30   CCCATGATCGTCCGAGTCTCGTGGGCTCGG   1um   STD      Round 1 blocking,CCCATGATCGTCCGAGTCTCGTGGGCTCGG,1um,STD      Round 2 blocking   30   ATCCACGTGCTTGAGCGCGCTGCATACTTG   1um   STD      Round 2 blocking,ATCCACGTGCTTGAGCGCGCTGCATACTTG,1um,STD      Round 3 blocking   30   GTGGCCGATGTTTCGCATCGGCGTACGACT   1um   STD      Round 3 blocking,GTGGCCGATGTTTCGCATCGGCGTACGACT,1um,STD     
 

SECTION: 1.2 Annealing Oligo plates - making oligo plates
6. Thaw deepwell plates at RT for 20-40 minutes at RT, and spin down before usage to avoid oligo cross-contamination. Can speed the thaw by setting on a thermomixer set to 28ºC if needed.
SECTION: 1.2 Annealing Oligo plates - making oligo plates
7. Make 50mL of STE, recipe below:
 
       Oligo Annealing buffer (STE)         Volume (mL)         1x Concentration            1M Tris pH 8.0         0.5         10 mM            5M NaCl         0.5         50 mM            0.5 M EDTA         0.1         1 mM            H2O         48.9                      Total         50                
 
SECTION: 1.2 Annealing Oligo plates - making oligo plates
8. Using a 12 channel multipipettor:
9. Dilute 180μl Round 1 linker oligo (1 mM) with 7920 μl STE buffer. Mix 80 μl diluted Round 1 linker oligo with 20 μl Round 1 oligo (100 μM) in a 96 well PCR plate.
SECTION: 1.3 Ordering and annealing oligos for cDNA tagmentation
18. SHARE-seq generates long cDNA (presuming no nuclease contamination or other harms befall the RNA).  In this state it is overlong to sequence, so we must tagment it using Tn5 complexed with Illumina Read 1 only.  To do this, we use commercial Tn5 from Diagenode, and incubate it with annealed oligos that include Illumina Read 1, and block the ME_Comp region of that sequence.
10. Dilute 259.2 μl Round 3 linker oligo (1 mM) with 7047 μl STE buffer. Mix 72 μl diluted Round 3 linker oligo with 28 μl Round 3 oligo (100 μM) in a 96 well PCR plate.
11. Dilute 216μl Round 2 linker oligo (1 mM) with 7371 μl STE buffer. Mix 76 μl diluted Round 2 linker oligo with 24 μl Round 2 oligo (100 μM) in a 96 well PCR plate.
12. Seal the plates - we use an BioRad PX1 plate sealer, seal at 180 degrees C for 5 seconds.   We use the specific foil seals for that sealer as well (part number 1814045).  If you don't have a heat sealer, we recommend BioRad Microseal F foil seals (part number MSF1001), which we use for sealing the final oligo plates as well.
13. Anneal Round 1, Round 2, and Round 3 plates using the PCR cycling conditions below.  The slow ramp is critical for this step!!  The plates won't work without the exact cycling conditions below!
 
       95ºC         2 min            Slow ramp, -1ºC/cycle,   1 min per cycle            20ºC         2 min            4ºC         Forever            Total         ~1h 26 min      
 
14. After annealing, check if there is significant water evaporation for the wells at the corners/sides of the wells.  With the PX1 heat sealing, we don't find that this occurs, but if you used more standard foil seals, there may be some evaporation on the sides.
15. Add 100μl STE to each well of the annealed plate, mix 7-8x and transfer 100μl to another 96 well plate.  These two plates are your "stock plates" from which you will aliquot into the multi-colored plates for each round of SHARE.
16. Aliquot 10ul from your stock plates (you'll have either 3 or 6 sets) to make an oligo plate.  We use the Agilent Bravo to make this go faster, but it's fairly quick with a 12 channel pipette as well.  Don't forget to: label the plates!  The rounds have to go in order for hybridization to work!  Also don't forget to put each "round" of oligo in a separate colored 96 well plate to reduce errors when selecting plates for the experiment.
17. Seal plates with foil and label using labels or marker (printed labels are faster if you've got them). Store the plates in rack at -30ºC, they are good for at least 6 months, and probably 12 months.
SECTION: 1.3 Ordering and annealing oligos for cDNA tagmentation
19. Order the following oligos from IDT resuspended at 100uM in IDTE
               
  Name
  LENGTH
  Sequence
  Scale
  Purification
  Specific oligos
  order from IDT
  
  Round 1 linker
  30
  CCGAGCCCACGAGACTCGGACGATCATGGG
  1um
  STD
  
  Round 1 linker,CCGAGCCCACGAGACTCGGACGATCATGGG,1um,STD
  
  Round 2 linker
  30
  CAAGTATGCAGCGCGCTCAAGCACGTGGAT
  1um
  STD
  
  Round 2 linker,CAAGTATGCAGCGCGCTCAAGCACGTGGAT,1um,STD
  
  Round 3 linker
  30
  AGTCGTACGCCGATGCGAAACATCGGCCAC
  1um
  STD
  
  Round 3 linker,AGTCGTACGCCGATGCGAAACATCGGCCAC,1um,STD
  
  Round 1 blocking
  30
  CCCATGATCGTCCGAGTCTCGTGGGCTCGG
  1um
  STD
  
  Round 1 blocking,CCCATGATCGTCCGAGTCTCGTGGGCTCGG,1um,STD
  
  Round 2 blocking
  30
  ATCCACGTGCTTGAGCGCGCTGCATACTTG
  1um
  STD
  
  Round 2 blocking,ATCCACGTGCTTGAGCGCGCTGCATACTTG,1um,STD
  
  Round 3 blocking
  30
  GTGGCCGATGTTTCGCATCGGCGTACGACT
  1um
  STD
  
  Round 3 blocking,GTGGCCGATGTTTCGCATCGGCGTACGACT,1um,STD
  
  Read1
  33
  TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
  1um
  HPLC
  *
  Read1,TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG,1um,HPLC
  
  RT_primer
  61
  /5Phos/GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGNNNNNNNNNN/iBiodT/TTTTTTTTTTTTTTVN
  1um
  RNASE
  *
  RT_primer,/5Phos/GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGNNNNNNNNNN/iBiodT/TTTTTTTTTTTTTTVN,1um,RNASE
  
  P7
  24
  CAAGCAGAAGACGGCATACGAGAT
  100nm
  STD
  *
  P7,CAAGCAGAAGACGGCATACGAGAT,100nm,STD
  
  Phosphorylated_Read2
  
  /5Phos/GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG
  1um
  HPLC
  *
  Phosphorylated_Read2,/5Phos/GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG,1um,HPLC
  
  Blocked_ME_Comp
  
  /5Phos/C*T*G* T*C*T* C*T*T* A*T*A* C*A*/3ddC/ 
  1um
  HPLC
  *
  Blocked_ME_Comp,/5Phos/C*T*G* T*C*T* C*T*T* A*T*A* C*A*/3ddC/
  ,1um,HPLC
    
Wait for them to arrive before proceeding!
SECTION: 1.3 Ordering and annealing oligos for cDNA tagmentation
20. 
Prepare tagmentation adapter mix in PCR tubes. 
 
  
  Tagmentation Adapter
  
  
  Volume (ml)
  
  
  
  100mM
  Read1
  
  
  26
  
  
  
  100mM
  Blocked_ME_Comp
  
  
  26
  
  
  
  1M Tris pH 8.0
  
  
  0.52
  
  
  
  5M NaCl
  
  
  0.52
  
  
  
  Total
  
  
  53
  
  

SECTION: 1.3 Ordering and annealing oligos for cDNA tagmentation
21. Anneal oligos in a thermal cycle as follows.
 
  
  85ºC
  
  
  2 min
  
  
  
  Slow ramp, -1ºC/cycle,
  1 min per cycle
  
  
  
  20ºC
  
  
  2 min
  
  
  
  4ºC
  
  
  Forever
  
  
  
  Total
  
  
  ~1h 14 min
  
  

SECTION: 1.3 Ordering and annealing oligos for cDNA tagmentation
22. Heat a 150ul aliquot of RNase free 100% glycerol to 65ºC in a heat block or thermomixer (RNase zap the surfaces).  Make two 50ul aliquots and equilibrate to RT.  Mix the annealed tagmentation adapter with an aliquot of glycerol, respectively (i.e. mix each 50ul aliquot of adapter with a separate 50ul aliquot of glycerol).  The annealed adapters can be immediately used, and stored at -20ºC for at least 6 months.
SECTION: 1.4 Essential SHARE-seq buffers (make the day before performing an experiment)
23. Make each of the following buffers - note that for large experiments you may need more than one aliquot of these buffers.

 
  
  Dilution buffer
  
  
  Volume (ml)
  
  
  Final Concentration
  
  
  
  100% glycerol (wide-bore tips)
  
  
  500
  
  
  50%
  
  
  
  1M Tris pH 7.5
  
  
  50
  
  
  50 mM
  
  
  
  5M NaCl
  
  
  20
  
  
  100 mM
  
  
  
  5mM EDTA
  
  
  20
  
  
  0.1 mM
  
  
  
  1M DTT
  
  
  1
  
  
  1 mM
  
  
  
  10% NP-40
  
  
  10
  
  
  0.1%
  
  
  
  H2O
  
  
  390
  
  
   
  
  
  
  Total
  
  
  1000
  
  
   
  
  

 
  
  5x SMART RT buffer 
  (adapted from smart-seq-3)
  
  
  Volume (ml)
  
  
  Final Concentration (of 5x)
  
  
  
  1000mM (1M) DTT
  
  
  40
  
  
  40 mM
  
  
  
  1M Tris pH 8.3
  
  
  125
  
  
  125 mM
  
  
  
  100 mM GTP
  
  
  50
  
  
  5 mM
  
  
  
  5M NaCl
  
  
  30
  
  
  150 mM
  
  
  
  1M MgCl2
  
  
  12.5
  
  
  12.5 mM
  
  
  
  H2O
  
  
  742.5
  
  
   
  
  
  
  Total
  
  
  1000
  
  
   
  
  

SECTION: 2.0 Washing and Counting Fixed cells or nuclei (time: 30-60m)
25. This section presumes cells or isolated nuclei have been fixed in formaldehyde (0.2% for cultured cells/nuclei, 1% for primary PBMCs) and have been frozen at -80 degrees as a dry pellet.  For more details on nuclei isolation, see the attached files (will be made into protocols.io links as time permits):
  #fixation and freezing guide for primary/cultured cells.
  #nuclear preparations from post-mortem tissue, includes an iodixanol gradient step and fixation instructions.

Important note: if your samplea are PBMCs or HSCs, you'll want to use the option polyAdenylation module (section 4) and do your centrifugation spins at 1000xg.

SECTION: 2.0 Washing and Counting Fixed cells or nuclei (time: 30-60m)
26. Put swinging bucket centrifuge rotor (Eppendorf # S-24-11-AT) in the refrigerable benchtop Eppendorf centrifuge (model #5430R) and cool down to 4ºC.
SECTION: 2.0 Washing and Counting Fixed cells or nuclei (time: 30-60m)
27. Prepare RNase free, sterile NIB-2RI, NI-2RI, NID-2RI (as well as NIB and NI)

 
  
  Nuclei Isolation
  Buffer (NIB)
  
  
  Volume (mL)
  
  
  Final Concentration
  
  
  
  1M Tris-HCl pH 7.5
  
  
  0.5
  
  
  10mM
  
  
  
  5M NaCl
  
  
  0.1
  
  
  10mM
  
  
  
  1M MgCl2
  
  
  0.15
  
  
  3mM
  
  
  
  10% NP40
  
  
  0.5
  
  
  0.1%
  
  
  
  H2O
  
  
  48.75
  
  
   
  
  
  
  Total
  
  
  50
  
  
   
  
  

 
  
  Nuclei Isolation (NI)
  
  
  Volume (mL)
  
  
  Final Concentration
  
  
  
  1M Tris-HCl pH 7.5
  
  
  0.5
  
  
  10mM
  
  
  
  5M NaCl
  
  
  0.1
  
  
  10mM
  
  
  
  1M MgCl2
  
  
  0.15
  
  
  3mM
  
  
  
  H2O
  
  
  49.25
  
  
   
  
  
  
  Total
  
  
  50
  
  
   
  
  
 
 
  
  NIB-2RI
  
  
  Volume (ml)
  
  
  
  NIB
  
  
  3000
  
  
  
  Enzymatic RI
  
  
  7
  
  
  
  SUPERase RI
  
  
  7
  
  

 
  
  NI-2RI
  
  
  Volume (ml)
  
  
  
  NI (per expt)
  
  
  18000
  
  
  
  Enzymatic RI
  
  
  45
  
  
  
  SUPERase RI
  
  
  45
  
  
  
  7.5% BSA
  
  
  96
  
  

SECTION: 2.0 Washing and Counting Fixed cells or nuclei (time: 30-60m)
28. Transfer fixed cells to a new 1.5mL tube coated with 7.5% BSA.  If starting from a fixed pellet, use 500ul NI-2RI to resuspend and transfer to a BSA coated tube.

Preparing 1.5ml BSA coated tubes: Prepare a 1.5mL Eppendorf tube by adding 150ml 7.5% BSA, vortex, remove supernatant, let tube sit on ice briefly, remove any collected BSA with
a P20.
SECTION: 2.0 Washing and Counting Fixed cells or nuclei (time: 30-60m)
29. Spin 750xg, 4ºC, 5 minutes. Gently remove supernatant.
SECTION: 2.0 Washing and Counting Fixed cells or nuclei (time: 30-60m)
30. Wash with 500ul NID-2RI without disturbing pellet.
SECTION: 2.0 Washing and Counting Fixed cells or nuclei (time: 30-60m)
31. Spin 750xg, 4ºC, 5 minutes. Gently remove supernatant.
SECTION: 2.0 Washing and Counting Fixed cells or nuclei (time: 30-60m)
32. Add 100ul NIB-2RI to each sample, incubate for 5 minutes on ice to permeabilize the nuclei.
SECTION: 2.0 Washing and Counting Fixed cells or nuclei (time: 30-60m)
35. Turn on a thermomixer (Eppendorf ThermoMixer C) for the ATAC Tn5 transposition, set to 37 degrees and put the 1.5mL block on the thermomixer (you should have a 96 well block for the hybridization step).



SECTION: 1.4 Essential SHARE-seq buffers (make the day before performing an experiment)
167. 
SECTION: 1.5 Ordering SHARE-seq oligos
24. All of the below oligos are necessary for performing SHARE-seq (mainly amplifying libraries on Day 2 of SHARE).  Wait until these arrive to start the SHARE-seq protocol.  All should be ordered formulated at 100uM in IDTE.
 
               
  Name
  LENGTH
  Sequence
  Scale
  Purification
  Specific oligos
  order from IDT
  
  Round 1 linker
  30
  CCGAGCCCACGAGACTCGGACGATCATGGG
  1um
  STD
  
  Round 1 linker,CCGAGCCCACGAGACTCGGACGATCATGGG,1um,STD
  
  Round 2 linker
  30
  CAAGTATGCAGCGCGCTCAAGCACGTGGAT
  1um
  STD
  
  Round 2 linker,CAAGTATGCAGCGCGCTCAAGCACGTGGAT,1um,STD
  
  Round 3 linker
  30
  AGTCGTACGCCGATGCGAAACATCGGCCAC
  1um
  STD
  
  Round 3 linker,AGTCGTACGCCGATGCGAAACATCGGCCAC,1um,STD
  
  Round 1 blocking
  30
  CCCATGATCGTCCGAGTCTCGTGGGCTCGG
  1um
  STD
  
  Round 1 blocking,CCCATGATCGTCCGAGTCTCGTGGGCTCGG,1um,STD
  
  Round 2 blocking
  30
  ATCCACGTGCTTGAGCGCGCTGCATACTTG
  1um
  STD
  
  Round 2 blocking,ATCCACGTGCTTGAGCGCGCTGCATACTTG,1um,STD
  
  Round 3 blocking
  30
  GTGGCCGATGTTTCGCATCGGCGTACGACT
  1um
  STD
  
  Round 3 blocking,GTGGCCGATGTTTCGCATCGGCGTACGACT,1um,STD
  
  Read1
  33
  TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
  1um
  HPLC
  *
  Read1,TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG,1um,HPLC
  
  RT_primer
  61
  /5Phos/GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGNNNNNNNNNN/iBiodT/TTTTTTTTTTTTTTVN
  1um
  RNASE
  *
  RT_primer,/5Phos/GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGNNNNNNNNNN/iBiodT/TTTTTTTTTTTTTTVN,1um,RNASE
  
  P7
  24
  CAAGCAGAAGACGGCATACGAGAT
  100nm
  STD
  *
  P7,CAAGCAGAAGACGGCATACGAGAT,100nm,STD
  
  Phosphorylated_Read2
  
  /5Phos/GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG
  1um
  HPLC
  *
  Phosphorylated_Read2,/5Phos/GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG,1um,HPLC
  
  Blocked_ME_Comp
  
  /5Phos/C*T*G* T*C*T* C*T*T* A*T*A* C*A*/3ddC/ 
  1um
  HPLC
  *
  Blocked_ME_Comp,/5Phos/C*T*G* T*C*T* C*T*T* A*T*A* C*A*/3ddC/
  ,1um,HPLC
  
  RNA_PCR_primer
  23
  AAGCAGTGGTATCAACGCAGAGT
  100nm
  STD
  *
  RNA_PCR_primer,AAGCAGTGGTATCAACGCAGAGT,100nm,STD
    

 
     
  Name
  LENGTH
  Sequence
  Scale
  Purification
  Specific oligos
  order from IDT
  
  TSO
  30
  AAGCAGTGGTATCAACGCAGAGTGAATrGrG+G
  1umR
  RNASE
  *
  TSO,AAGCAGTGGTATCAACGCAGAGTGAATrGrG+G,1umR,RNASE
    

 
SECTION: 2.0 Washing and Counting Fixed cells or nuclei (time: 30-60m)
34. Spin 750xg, 4ºC, 5 minutes. Gently remove supernatant, using a P20 to remove as much NIB-2RI as possible.
SECTION: 2.0 Washing and Counting Fixed cells or nuclei (time: 30-60m)
36. Resuspend in 50ul HCB-RI (400ul 1xHCB + 1ul Enzymatic RI) or NI-RI (same recipe using NI).  SUPERase RI inhibits the Tn5 transposition, so using RI instead of 2RI is very important here.
SECTION: 2.0 Washing and Counting Fixed cells or nuclei (time: 30-60m)
37. Use 2.5ul cells and 7.5ul HCB-RI plus 10ul Trypan blue to count nuclei using the BioRad TC20 (part number 1450102) or a manual hemocytometer (we like the Reichert Bright-Line: http://hausserscientific.com/products/reichert_bright_line.html).  Determine cell counts per mL and per ul, so the volume needed to get to 10000 nuclei is known.  Multiply the count by 4, since the cells/nuclei are diluted 1:4 before adding trypan blue.  The TC20 takes trypan blue dilutions into account, so divide the count per mL by 1000 to get the count per ul.
SECTION: 2.0 Washing and Counting Fixed cells or nuclei (time: 30-60m)
38. Determine the volume of cell suspension that is 10,000 cells (one ATAC transposition), and the total number of ATAC reactions (10,000 cells/reaction).  We typically do SHARE using 25 ATAC reactions (250K cells) as the basic "unit" of SHARE.


SECTION: 3.0 ATAC Tn5 transposition (time: ~45m)
39. Assemble transposome by mixing assembled Tn5, and dilution buffer.  Here we use volumes of ASSEMBLED Tn5 from the SeqWell produced Tn5 that the Buenrostro lab also uses.
SECTION: 3.0 ATAC Tn5 transposition (time: ~45m)
40. 
 
  
  Transposome
  
  
  Volume (ml)
  
  
  Volume N=25
  
  
  
  1x SeqWell Tn5
  (volume as of 7/27/2021)
  
  
  0.6 ✕
  N reactions
  
  
  15ml
  
  
  
  Dilution buffer
  
  
  1.875 ✕
  N reactions
  
  
  46.9ml
  
  
  
  Total
  
  
  2.5 ✕
  N reactions
  
  
  61.9ml
  
  

 
 
SECTION: 3.0 ATAC Tn5 transposition (time: ~45m)
39.1. We use 0.6ul per reaction (10000 cells) (enzyme concentration is 10mM).
SECTION: 3.0 ATAC Tn5 transposition (time: ~45m)
41. Calculate the total number of ATAC reactions for this experiment.  Prepare 1x TB accordingly to the recipe below. Note that 1x TB buffer must be made fresh. 
 
  
  1x TB
  
  
  Volume (ml) ✕
  samples (N)
  
  
  Volume N=27.5 (per tube plus overage)
  
  
  
  0.2M Tris-acetate
  
  
  8.25 ✕
  N
  
  
  226.875
  
  
  
  5M K-acetate
  
  
  0.66 ✕
  N
  
  
  18.15
  
  
  
  1M Mg-acetate
  
  
  0.5 ✕
  N
  
  
  13.75
  
  
  
  100% DMF
  
  
  8 ✕ N
  
  
  220
  
  
  
  H2O
  
  
  24.04 ✕
  N
  
  
  661.1
  
  
  
  PIC
  
  
  0.2 ✕
  N
  
  
  5.5
  
  
  
  Enzymatic RI
  
  
  0.85 ✕ N
  
  
  23.375
  
  
  
  Total
  
  
            42.5 ✕ N
  
  
  1168.75
  
  

SECTION: 3.0 ATAC Tn5 transposition (time: ~45m)
42. The total volume of cells for the N number of ATAC reactions should fit into a 50ul reaction.  Determine the total volume of cells needed for the ATAC reactions, and then use NI to bring the total volume up to 50ul.  Example: with 25 ATAC reactions (250K cells), and 13,920 cells per ul, use 250K/13920 to get 17.95ul of cells needed. Add 50ul-14.4ul = 32.05ul NI-RI to get 50ul total volume of cells.
   
SECTION: 3.0 ATAC Tn5 transposition (time: ~45m)
43. Add 42.5 ✕ N ul of 1xTB to cells + NI (in 50ul total volume): 1062.5ul for 25 rxns/sample, incubate at RT for 10 minutes (this is a permeabilization step).
SECTION: 3.0 ATAC Tn5 transposition (time: ~45m)
44. Add 2.5 ✕ N ul (61.9ul for 25 reactions) of assembled Tn5 to sample, mix well by pipetting.



SECTION: 3.0 ATAC Tn5 transposition (time: ~45m)
45. In the 1.5mL tube, seal, shake in a thermomixer at 500rpm for 30 minutes, 37ºC.  
SECTION: 3.0 ATAC Tn5 transposition (time: ~45m)
45.1. Every 5 minutes, take the entire block off the thermomixer and invert to mix
This resets the timer on the thermomixer!  So use an external timer
SECTION: 3.0 ATAC Tn5 transposition (time: ~45m)
46. Add 500ul NID-2RI to each tube, spin down at 750xg, RT, 5 minutes.
SECTION: 3.0 ATAC Tn5 transposition (time: ~45m)
47. Remove supernatant, wash with 500ul NI-2RI, spin down at 750xg, RT, 5 minutes.


SECTION: 3.0 ATAC Tn5 transposition (time: ~45m)
48. Remove supernatant, resuspend cells in 60ul NI-2RI (from a single ATAC reaction tube).
SECTION: 3.0 ATAC Tn5 transposition (time: ~45m)
49. Set thermomixer(s) to 23ºC for the hybridization reaction (after RT) and put the 96 well blocks on.
SECTION: 5.0 Reverse Transcription (time: 50m-1h)
57. Make the following master mix for reverse transcription:

 
  
  Reverse
  transcription (RT) mix
  
  
  Volume/sample
  (ml)
  
  
  
  5x SMART RT buffer
  
  
  70
  
  
  
  Enzymatic RI
  
  
  2.19
  
  
  
  SUPERase RI
  
  
  4.38
  
  
  
  dNTPs
  
  
  17.5
  
  
  
  100mM RT primer
  
  
  35
  
  
  
  H2O
  
  
  40.94
  
  
  
  50% PEG
  
  
  35
  
  
  
  Maxima H Minus Reverse Transcriptase (add
  right before RT reaction)
  
  
  35
  
  
  
  Total
  
  
  240
  
  


For each ATAC 25 reaction tube, add 280ul RT mix to 60ul cells/NI-2RI, aliquot 50ul to 7 wells in a 96 well plate.  Then, run the RT thermocycler protocol as described below.
 
 
  
  50ºC
  
  
  10 min
  
  
   
  
  
  
  8ºC
  
  
  12 s
  
  
  3 cycles
  
  
  
  15ºC
  
  
  45 s
  
  
  
  20ºC
  
  
  45 s
  
  
  
  30ºC
  
  
  30 s
  
  
  
  42ºC
  
  
  2 min
  
  
  
  50ºC
  
  
  3 min
  
  
  
  50ºC
  
  
  5 min
  
  
   
  
  
  
  Total
  
  
  ~41 min
  
  
   
  
  

SECTION: 5.0 Reverse Transcription (time: 50m-1h)
58. Pool all reactions for a given sample, add 300ul NID-2RI per 7 PCR tubes, spin down at 750xg, 5minutes, RT in a 1.5mL Eppendorf tube coated with 7.5% BSA. Note: the pellet may look larger than before - this is due to BSA dropping out of solution.
SECTION: 5.0 Reverse Transcription (time: 50m-1h)
59. Wash with 500ul NI-2RI, spin down at 750xg, 5minutes, RT
SECTION: 6.0 Hybridization
60. Remove supernatant and resuspend each sample in NI-2RI.  The amount each sample is resuspended in depends on the number of samples, and plates, per experiment.  For a single sample in a single plate (96 well): resuspend in 1152ul NI-2RI.  For four samples on a single plate, resuspend each sample in 288ul NI-2RI.


 

SECTION: 6.0 Hybridization
61. Note: it is very important to thaw oligo plates to RT (~23ºC) before hybridization.  Spin down plates before using.  Coat all reservoirs used for pooling with 7.5% BSA.

Make the hybridization buffer:

 
  
  Hybridization buffer
  
  
  Volume (ml)
  
  
  Stock concentration
  
  
  Final Concentration
  
  
  
  H2O
  
  
  5510
  
  
  --
  
  
  --
  
  
  
  T4 ligase buffer
  (10x)
  
  
  1152
  
  
  10x
  
  
  1x
  
  
  
  Enzymatic RI
  
  
  28.8
  
  
  20 U/ml
  
  
  0.05 U/ml
  
  
  
  SUPERase RI
  
  
  92.16
  
  
  40 U/ml
  
  
  0.32 U/ml
  
  
  
  7.5% BSA
  
  
  184.32
  
  
  7.5%
  
  
  0.1%
  
  
  
  Total
  
  
  6912
  
  
   
  
  
   
  
  

 
           
  Hybridization
  buffer
  Volume (ml)
  Stock concentration
  Final Concentration
  
  H2O
  2755
  --
  --
  
  T4
  ligase buffer (10x)
  576
  10x
  1x
  
  Enzymatic
  RI
  14.4
  20 U/ml
  0.05 U/ml
  
  SUPERase
  RI
  46.08
  40 U/ml
  0.32 U/ml
  
  7.5% BSA
  92.16
  7.50%
  0.10%
  
  Total
  3483.64
   
   
    


SECTION: 6.0 Hybridization
62. If you have a single sample: transfer the cells in NI-2RI into a BSA-coated reservoir suitable for an 8 or 12 channel multi-channel pipette.   Add all of the hybridization buffer to the reservoir (adjust volume for 1 or 2 plates).
SECTION: 6.0 Hybridization
65. Transfer sample(s) to Round 1 plate(s) as follows for each row of wells:
a.	Set a 12-channel pipette to 45ul, mix 2-3x 
b.	Transfer 45ul of mixture to Round 1 plates and mix by pipetting gently.
c.	Change tips between columns loaded!
SECTION: 6.0 Hybridization
66. Seal and shake at 300rpm for 25 minutes at RT (23 deg C).
SECTION: 6.0 Hybridization
64. We find that using a 96 well plate map helps a lot for sample mapping!  An example image is below, along with an open-access svg file of a 96 well plate.

 
      
For those who have access to photoshop or Gimp, the *.psd files are editable templates.  We recommend a making a layer to color the wells by sample identity.
SECTION: 6.0 Hybridization
67. Prepare Blocking oligo 1 as below and transfer to reservoir.
 
 
 
 

 
SECTION: 6.0 Hybridization
68. Add blocking oligo mix as follows:
a.	Using a 12-channel pipette, transfer 10ul Blocking oligo 1 to each well, pipetting up and down to mix thoroughly, changing tips between rows.
b.	Seal and shake at 300rpm for 15 minutes at RT
SECTION: 6.0 Hybridization
69. Pool samples as follows:
a.	Set a 12-channel pipette to 70ul, pipette each row up and down 2-3 times, pipette up and down 2-3 times as ejecting each row into a BSA-coated reservoir (avoid bubbles).
b.	After all rows have been pooled, pipette up and down 4-5 times to mix the reservoir
SECTION: 6.0 Hybridization
70. Open Round 2 oligo plates, and transfer 55ul of pooled mixture to each well, pipetting to mix each row.  Shake at 300rpm for 25 minutes at RT.
SECTION: 6.0 Hybridization
71. Prepare Blocking oligo 2 as below and transfer to reservoir.
 
 
 
 

SECTION: 6.0 Hybridization
72. Add blocking oligo mix as follows:
a.	Using a 12-channel pipette, transfer 10ul Blocking oligo 2 to each well, pipetting up and down to mix thoroughly, changing tips between rows.
b.	Seal and shake at 300rpm for 15 minutes at RT
SECTION: 6.0 Hybridization
73. Pool samples as follows:
a.	Set a 12-channel pipette to 80ul, pipette each row up and down 2-3 times, pipette up and down 2-3 times as ejecting each row into a BSA-coated reservoir (avoid bubbles).
b.	After all rows have been pooled, pipette up and down 4-5 times to mix the reservoir
SECTION: 6.0 Hybridization
74. Open Round 3 oligo plate, and transfer 65ul of pooled mixture to each well, pipetting to mix each row.  Shake at 300rpm for 25 minutes at RT.



SECTION: 6.0 Hybridization
75. Prepare Blocking oligo 3 as below and transfer to reservoir.
 
 
 

SECTION: 6.0 Hybridization
76. Add blocking oligo mix as follows:
a.	Using a 12-channel pipette, transfer 10ul Blocking oligo 3 to each well, pipetting up and down to mix thoroughly, changing tips between rows.
b.	Seal and shake at 300rpm for 15 minutes at RT.
SECTION: 7.0 Ligation (time: ~45m)
77. Using either a P1000, or a 12-channel pipette and a BSA coated reservoir, aspirate the contents of each well and pool into a 15mL conical tube coated with 7.5% BSA.
a.	Total volume should be ~14mL (for a two plate experiment), but will fit in a single tube.
b.     For a single plate experiment, the totla volume would be 7mL
SECTION: 7.0 Ligation (time: ~45m)
78. Spin down at 750xg for 5 minutes at RT in a swinging bucket rotor.
SECTION: 7.0 Ligation (time: ~45m)
79. Discard supernatant and add 1mL NID-2RI.
SECTION: 7.0 Ligation (time: ~45m)
80. Spin down at 750xg for 5 minutes at RT in a swinging bucket rotor.
SECTION: 7.0 Ligation (time: ~45m)
81. Discard supernatant and add 1mL NI-2RI, without disturbing pellet (let flow down side of tube slowly)
SECTION: 7.0 Ligation (time: ~45m)
82. Spin down at 750xg for 5 minutes at RT in a swinging bucket rotor.
SECTION: 7.0 Ligation (time: ~45m)
83. Resuspend in 80ul NI-2RI.
SECTION: 7.0 Ligation (time: ~45m)
84. Mix sample with 320ul ligation mix (recipe below) and aliquot 50ul to 8 PCR tubes.

 
 
  
  Ligation mix
  
  
  Volume (ml)
  
  
  
  H2O
  
  
  253.7
  
  
  
  Enzymatic RI
  
  
  3.2
  
  
  
  SUPERase RI
  
  
  1
  
  
  
  T4 ligase buffer
  (10x)
  
  
  40
  
  
  
  T4 ligase 400U /ml
  
  
  20
  
  
  
  7.5% BSA
  
  
  2.13
  
  
  
  Total
  
  
  320
  
  

SECTION: 7.0 Ligation (time: ~45m)
85. Shake at 300rpm for 30 min at RT.
SECTION: 7.0 Ligation (time: ~45m)
86. Pool samples in a 1.5mL Eppendorf tube coated with 7.5% BSA, spin down 750xg, 5 minutes, RT, discard supernatant.
SECTION: 7.0 Ligation (time: ~45m)
87. Gently resuspend pellet with 1mL NI-2RI
SECTION: 7.0 Ligation (time: ~45m)
88. Spin at 750xg, 5 minutes, RT.
SECTION: 7.0 Ligation (time: ~45m)
89. Presume 80% loss of nuclei over the course of the protocol, so for every 20,000 retained nuclei, resuspend in 10ul (goal is ~2000 cells or nuclei/ul).  
For example: With 1 million starting cells, presume 100K are retained, and resuspend in 50ul NI-2RI.
SECTION: 7.0 Ligation (time: ~45m)
90. Using 2ul cells and 8ul NI-2RI, mix with 10ul trypan blue.  Count intact nuclei in a manual hemocytometer.  
To get a count per mL, multiply hemocytometer counts by 100K 
(dilution factors: 2x = trypan blue, 5x = cell:NI dilution, 10,000x = hemocytometer dilution).
To get a count per ul, multiply counts by 100 (conversion: 100 = 100,000 (dilution factor for hemocytometer plus dilutions)/1,000 (microliters in one mL)).
SECTION: 8.0 Reverse crosslinking-pull down (time: 1h15m)
91. Here you will aliquot cells into cellular or nuclear "sub pools", and then digest the remaining proteins away to leave only barcoded nucleic acids.  The number of cells per pool strongly depends on the experimental setup: a 96 barcode (single plate) experiment should not contain more than 25,000 cells or nuclei - the risk of barcode collisions (two different cells with the same randomly generated barcode) increases substantially (>2.5%).  

A 192 barcode (two plate) experiment has a maximum cell count of 100,000 cells per cellular or nuclear sub pool, though theoretically 200,000 should be possible (however we have not tested cellular/nuclear sub pools of this size at present 01/2024).  Other important points:  nuclease rich cells benefit from extra RNase inhibitors at the proteinase K stage as well as on the second day of SHARE.  We have increased these numbers to working with pools around 50,000-100,000 cells or nuclei, but you may need to adjust these numbers depending on your source material.  Replicates are also important: if you return 50,000 cells or nuclei for a two plate experiment, doing two cellular/nuclear sub pools of 25,000 cells is better than a single pool - it allows for concordance checking across replicates as well as providing some insurance for handling samples on the second day of SHARE.
SECTION: 8.0 Reverse crosslinking-pull down (time: 1h15m)
92. Bring the volume of each sub-library up to 50ul using NI-2RI
SECTION: 8.0 Reverse crosslinking-pull down (time: 1h15m)
93. For proteinase K digestion, add 50ul 2x RCB, 2ul proteinase K (20mg/mL), 2ul SUPERase RI

 
  
  2x RCB
  
  
  Volume (mL)
  
  
  2x Concentration
  
  
  
  1M Tris pH 8.0
  
  
  1
  
  
  100 mM
  
  
  
  5M NaCl
  
  
  0.2
  
  
  100 mM
  
  
  
  20% SDS
  
  
  0.2
  
  
  0.40%
  
  
  
  H2O
  
  
  8.58
  
  
   
  
  
  
  Total
  
  
  10
  
  
   
  
  

 
 
 
  
  ProtK mix
  
  
  Volume (ul) ✕ samples (N+1)
  
  
  5 samples: N+1=6
  
  
  10 samples N+1=11
  
  
  20 samples N+1=21
  
  
  
  RCB
  
  
  25
  
  
  150
  
  
  275
  
  
  525
  
  
  
  Proteinase K
  (20mg/mL)
  
  
  2
  
  
  12
  
  
  22
  
  
  42
  
  
  
  SUPERase RI
  
  
  2
  
  
  12
  
  
  22
  
  
  42
  
  
  
  Cell
  in NI-2RI
  
  
  25
  
  
   
  
  
   
  
  
   
  
  
  
  Total
  
  
  53
  
  
  168
  
  
  308
  
  
  588
  
  

SECTION: 8.0 Reverse crosslinking-pull down (time: 1h15m)
94. Incubate at 55ºC for one hour on the thermal cycler with lid is set to 65ºC
SECTION: 8.0 Reverse crosslinking-pull down (time: 1h15m)
95. Can stop and store proteinase K digestions at -80ºC for up to a week.
SECTION: 8.0 Reverse crosslinking-pull down (time: 1h15m)
96. If coming from -80ºC, thaw samples on ice first. Mix each reaction with 2.5ul 100mM PMSF in IPA and incubate at RT for 10 minutes.
SECTION: 8.0 Reverse crosslinking-pull down (time: 1h15m)
97. Prepare the following buffers:
 
  
  2x BW
  
  
  Volume (mL)
  
  
  2x Concentration
  
  
  
  1M Tris pH 8.0
  
  
  0.5
  
  
  10 mM
  
  
  
  5M NaCl
  
  
  20
  
  
  2 M
  
  
  
  0.5M EDTA
  
  
  0.1
  
  
  1 mM
  
  
  
  H2O
  
  
  29.4
  
  
   
  
  
  
  Total
  
  
  50
  
  
   
  
  

 
  
  1x BW-T
  
  
  Volume (mL)
  
  
  1x Concentration
  
  
  
  1M Tris pH 8.0
  
  
  0.25
  
  
  5 mM
  
  
  
  5M NaCl
  
  
  10
  
  
  1 M
  
  
  
  0.5M EDTA
  
  
  0.05
  
  
  0.5 mM
  
  
  
  10% Tween 20
  
  
  0.25
  
  
  0.05%
  
  
  
  H2O
  
  
  39.675
  
  
   
  
  
  
  Total
  
  
  50
  
  
   
  
  
 
 
  
  Oligo Annealing buffer (STE)
  
  
  Volume (mL)
  
  
  1x Concentration
  
  
  
  1M Tris pH 8.0
  
  
  0.5
  
  
  10 mM
  
  
  
  5M NaCl
  
  
  0.5
  
  
  50 mM
  
  
  
  0.5 M EDTA
  
  
  0.1
  
  
  1 mM
  
  
  
  H2O
  
  
  48.9
  
  
   
  
  
  
  Total
  
  
  50
  
  
   
  
  

SECTION: 8.0 Reverse crosslinking-pull down (time: 1h15m)
96.1. To make 1mL PMSF in 100% IPA, use 17mg PMSF and weigh into a 1.5mL Eppendorf tube.


SECTION: 8.0 Reverse crosslinking-pull down (time: 1h15m)
98. Now prepare the above buffers with RNase inhibitors.
 
  
  2x
  BW/RI
  
  
  Volume/ul
  
  
  
  2x BW
  
  
  110x(N+1)
  
  
  
  SUPERase RI
  
  
  2x(N+1)
  
  

 
 
  
  1x
  BW-T/RI
  
  
  Volume/ul
  
  
  
  1x BW-T
  
  
  500x(N+1)
  
  
  
  SUPERase RI
  
  
  10x(N+1)
  
  

 
 
  
  1x
  STE/RI
  
  
  Volume/ul
  
  
  
  STE
  
  
  200x(N+1)
  
  
  
  SUPERase RI
  
  
  N+1
  
  

SECTION: 8.0 Reverse crosslinking-pull down (time: 1h15m)
99. In a separate tube, mix 10xN ul of MyOne C1 Dynabeads with 100xN ul of 1xBW-T and place on a magnetic rack
SECTION: 8.0 Reverse crosslinking-pull down (time: 1h15m)
100. Remove supernatant, wash twice with 100xN ul BW-T without RI
SECTION: 8.0 Reverse crosslinking-pull down (time: 1h15m)
101. Remove supernatant, wash once with 100xN ul BW-T/RI, and resuspend beads in 110xN ul 2x BW/RI.
SECTION: 8.0 Reverse crosslinking-pull down (time: 1h15m)
102. Add 100ul of washed beads to each sample, nutate at RT for 60 minutes
SECTION: 8.0 Reverse crosslinking-pull down (time: 1h15m)
103. Put on a magnetic rack, transfer supernatant containing transposed ATAC chromatin fragments to a new tube for future library preparation.  The supernatant is stable at RT for several hours (but we prepare the ATAC libraries right away,using a paired system where one person handles the cDNA, and the other can handle the ATAC, see step #102 below).
SECTION: 8.0 Reverse crosslinking-pull down (time: 1h15m)
104. Wash cDNA/RNA-bound beads with 100ul 1xBW-T/RI, three times.
SECTION: 8.0 Reverse crosslinking-pull down (time: 1h15m)
105. Place sample on magnetic rack, wash with 100ul 1xSTE/RI, without resuspending beads
SECTION: 9.0 RNA library preparation: template switching (time: 2h, ATAC prep during incubations)
106. Make the template switch mix using the recipe below:
 
 
 
  
  Template switch mix
  
  
  Volume (ml)
  
  
  Final Concentration
  
  
  
  H2O
  
  
  1.25x(N+1)
  
  
   
  
  
  
  50% PEG 6000
  
  
  15x(N+1)
  
  
  15%
  
  
  
  5x SMART RT buffer
  
  
  10x(N+1)
  
  
  1x
  
  
  
  Ficoll PM-400 (20%)
  
  
  10x(N+1)
  
  
  4%
  
  
  
  10 mM dNTPs, each
  
  
  5x(N+1)
  
  
  1 mM
  
  
  
  RNase inhibitor (Lucigen)
  
  
  10x(N+1)
  
  
  0.2x
  
  
  
  100 mM TSO
  
  
  1.25x(N+1)
  
  
  2.5 mM
  
  
  
  Maxima H Minus Reverse Transcriptase (add right
  before RT reaction)
  
  
  2.53x(N+1)
  
  
   
  
  
  
  Total
  
  
  50x(N+1)
  
  
   
  
  

SECTION: 9.0 RNA library preparation: template switching (time: 2h, ATAC prep during incubations)
107. Remove all supernatant from the beads, resuspend beads in 50ul Template switch mix, be careful to avoid beads drying out after removing supernatant
SECTION: 9.0 RNA library preparation: template switching (time: 2h, ATAC prep during incubations)
108. Rotate/nutate samples at RT for 30 minutes
SECTION: 9.0 RNA library preparation: template switching (time: 2h, ATAC prep during incubations)
109. Incubate at 42ºC for 90 minutes, shaking at 300rpm, resuspend beads at start, 30 minutes, 60 minutes, and at the end of the incubation by flicking the tubes to mix.  We find RNase contamination can happen when the samples are pipetted, so we recommend mixing the settled beads by flicking for this reason.
SECTION: 10.0 ATAC library preparation (time: 2h50m, variable depending on qPCR results)
110. Using the supernatnant with ATAC fragments from step 95, purify the samples using a Qiagen MinElute PCR purification kit (Qiagen cat #: 28006) as follows:
SECTION: 10.0 ATAC library preparation (time: 2h50m, variable depending on qPCR results)
110.1. In new tube, mix sample and 5x volume Qiagen PB buffer (1000ul), then add to MinElute spin column in two batches 2x600ul, spinning, then discarding flowthrough after each addition (as below).  
SECTION: 10.0 ATAC library preparation (time: 2h50m, variable depending on qPCR results)
110.2. Spin down the tube at 13,000rpm for 1 minute and discard the flowthrough 
SECTION: 10.0 ATAC library preparation (time: 2h50m, variable depending on qPCR results)
110.3. Add 750ul of PE Wash Buffer to the column, spin down at 13,000rpm for 1 minute, and discard flowthrough. 
SECTION: 10.0 ATAC library preparation (time: 2h50m, variable depending on qPCR results)
110.4. Spin the samples at 13,000rpm for 1 minute to capture any excess buffer.
SECTION: 10.0 ATAC library preparation (time: 2h50m, variable depending on qPCR results)
110.5. Place the spin column into a new tube and add 11ul of EB (10mM Tris, pH 8.0) directly to the column matrix. Incubate for 1 minute. 
SECTION: 10.0 ATAC library preparation (time: 2h50m, variable depending on qPCR results)
110.6. Spin down the tube at 13,000rpm for 1 minute to elute the DNA.
SECTION: 10.0 ATAC library preparation (time: 2h50m, variable depending on qPCR results)
110.7. Add 11ul EB to spin column, incubate for 1 minute, and spin again for total of ~20ul eluate. 
SECTION: 10.0 ATAC library preparation (time: 2h50m, variable depending on qPCR results)
111. Mix ~20ul sample with 29ul PCR mix (recipe below), 1ul of 25uM IDT8_i5 01-96 primer and H2O, run PCR for 5 cycles (as below)
 
 
  
  ATAC PCR temp
  
  
  Time
  
  
  Cycles
  
  
  
  72ºC
  
  
  5 min
  
  
  1 cycle
  
  
  
  98ºC
  
  
  30 s
  
  
  1 cycle
  
  
  
  98ºC
  
  
  10 s
  
  
  5 cycles
   
  ~22 min
  
  
  
  65ºC
  
  
  30 s
  
  
  
  72ºC
  
  
  60 s
  
  

 

 
 
SECTION: 10.0 ATAC library preparation (time: 2h50m, variable depending on qPCR results)
112. qPCR: using 5ul sample and 10ul qPCR mix (NEBnext, Ad1.xx, P7 primer, recipe below), run the qPCR cycle program below, and calculate the number of cycles needed to each 1/3 of the plateau fluorescence (0.33 Ct).  Don't forget to use a no template control (NTC) to check for PCR mastermix contamination!
 
 
  
  ATAC qPCR mix
  
  
  Volume (ul)
  
  
  
  NEBnext
  2x PCR mastermix
  
  
  5x(N+1)
  
  
  
  Ad1.01
  25uM
  
  
  0.2x(N+1)
  
  
  
  P7 primer
  25uM
  
  
  0.2x(N+1)
  
  
  
  10x
  SYBRgreen
  
  
  0.9x(N+1)
  
  
  
  H2O
  
  
  3.7x(N+1)
  
  
  
  Total
  
  
  10x(N+1)
  
  

 
 
  
  qPCR
  
  
  cycles
  
  
  
  98ºC
  
  
  30 s
  
  
  1 cycle
  
  
  
  98ºC
  
  
  10 s
  
  
  20 cycles
   
  ~1 hour
  
  
  
  65ºC
  
  
  30 s
  
  
  
  72ºC
  
  
  60 s
  
  

SECTION: 10.0 ATAC library preparation (time: 2h50m, variable depending on qPCR results)
113. Save the qPCR plate to check qPCR product size on an e-gel.  This can be combined with checking the cDNA after amplification. Expect a smear with an average size of 400bp.  Nucleosomal banding may be present.  A smaller MW band at ~270bp is likely non-nucleosomal, and does not correlate with poor quality.


[add image of qPCR gel for ATAC]

SECTION: 10.0 ATAC library preparation (time: 2h50m, variable depending on qPCR results)
114. Run additional PCR cycles for the rest of the sample (45ul), using the qPCR cycle schema above, where N=0.33 Ct cycles.  The number of additional cycles expected is complex and based on cell type as well as cell number.  Generally libraries with a large number of cells (25000-50000+) will need only 2-4 additional cycles of amplification.  Smaller numbers of cells (2000-10000) will need more like 6-10 additional cycles of amplification.  These are generalities, your samples may vary.

[add image of qPCR cycles for ATAC]
SECTION: 10.0 ATAC library preparation (time: 2h50m, variable depending on qPCR results)
115. After PCR reamp, add 1ul of Exonuclease I (NEB cat # M0293L) to each library and place libraries back in thermocycler for “ExoCleanup” program (parameters below) to get rid of excess primer.

 
 
  
  ExoCleanup
  
  
  
  37ºC
  
  
  15 mins
  
  
  
  80ºC
  
  
  15 mins
  
  
  
  4ºC
  
  
  Forever
  
  
  
  Total
  
  
  ~30 min
  
  

SECTION: 10.0 ATAC library preparation (time: 2h50m, variable depending on qPCR results)
116. Clean sample using Qiagen MinElute DNA clean up kit (225ul binding buffer, 250ul if qPCR stage was skipped), elute in 6ul EB, then repeat elution with 6ul EB, for a total of ~10ul eluate.  Libraries can be stored at  -20ºC or -80ºC.
SECTION: 11.0 cDNA amplification and cleanup (time 3h30m)
117. Mix each cDNA sample with 100ul H2O, magnetize and discard supernatant, wash with 200ul STE; don’t resuspend beads.
SECTION: 11.0 cDNA amplification and cleanup (time 3h30m)
118. Mix 50ul PCR mix with beads, keep in existing tubes

 
  
  cDNA PCR mix
  
  
  Volume (ul)
  
  
  
  Kapa Hifi 2x mastermix
  
  
  25x(N+1)
  
  
  
  RNA PCR primer 25uM
  
  
  0.8x(N+1)
  
  
  
  P7 primer 25uM
  
  
  0.8x(N+1)
  
  
  
  H2O
  
  
  23.4x(N+1)
  
  
  
  Total
  
  
  50x(N+1)
  
  

SECTION: 11.0 cDNA amplification and cleanup (time 3h30m)
119. Run PCR for 5 cycles
 
  
  PCR
  
  
  Cycles
  
  
  
  95ºC
  
  
  3 min
  
  
   
  
  
  
  98ºC
  
  
  20 s
  
  
  5 cycles
   
  ~32 min
  
  
  
  65ºC
  
  
  45 s
  
  
  
  72ºC
  
  
  3 min
  
  

SECTION: 11.0 cDNA amplification and cleanup (time 3h30m)
120. With samples in magnetic rack, transfer supernatant to new PCR wells.
SECTION: 11.0 cDNA amplification and cleanup (time 3h30m)
121. qPCR: for each sub-library, mix 2.5ul sample with 7.5ul qPCR mix, run qPCR protocol below and calculate 0.33 Ct. 
 
 
 
  
  cDNA qPCR mix
  
  
  Volume (ul)
  
  
  
  Kapa Hifi 2x mix
  
  
  3.75x(N+1)
  
  
  
  RNA PCR primer 25mM
  
  
  0.12x(N+1)
  
  
  
  P7 primer 25mM
  
  
  0.12x(N+1)
  
  
  
  EVAgreen 20x
  
  
  0.5x(N+1)
  
  
  
  H2O
  
  
  3.01x(N+1)
  
  
  
  Total
  
  
  7.5x(N+1)
  
  

 
  
  qPCR
  
  
   
  
  
  
  95ºC
  
  
  3 min
  
  
   
  
  
  
  98ºC
  
  
  20 s
  
  
  20
  cycles
   
  ~1h 38m
  
  
  
  65ºC
  
  
  20 s
  
  
  
  72ºC
  
  
  3 min
  
  

SECTION: 11.0 cDNA amplification and cleanup (time 3h30m)
122. Run additional PCR cycles for the rest of the sample (~50ul), N=0.33 Ct cycles, see step 106 above.  Cycle timing for cDNA is as multifaceted as ATAC, but has the additional complexity of possible nuclease degradation.   Generally, the more cells, the lower the cycle number, but this is highly variable across input material.  For cultured cell lines, we often see a 50,000+ cell sample will require [6-8] additional cycles of amplification.  This increases substantially for PBMCs and hematopoietic compartment cells especially - even for samples with 50,000 cells or more, we still generally require at least 10 cycles of amplification.
SECTION: 11.0 cDNA amplification and cleanup (time 3h30m)
123. Purify each sample (47.5ul) with 38ul AMpure beads (0.8x, equilibrated to RT) for cell line samples, or 28.5ul AMpure beads (0.6x) for primary samples.  Elute cDNA to 10ul EB (10 mM Tris, pH 8.0).  Can stop and store samples at -80ºC here.  Ampure XP protocol is below, as substeps:
SECTION: 11.0 cDNA amplification and cleanup (time 3h30m)
123.1. Bind samples and beads for 5 minutes at RT
SECTION: 11.0 cDNA amplification and cleanup (time 3h30m)
123.2. Magnetize for 5 minutes
SECTION: 11.0 cDNA amplification and cleanup (time 3h30m)
123.3. Wash 2x with 200ul fresh 80% ethanol with samples on magnet (~30s incubate with ethanol on beads)
SECTION: 11.0 cDNA amplification and cleanup (time 3h30m)
123.4. Dry for ~5 minutes (can place in fume hood to speed things up).  Don’t let the beads look cracked, they should look glossy, and just at the point of turning matte.
SECTION: 11.0 cDNA amplification and cleanup (time 3h30m)
123.5. Resuspend in 10ul EB
SECTION: 11.0 cDNA amplification and cleanup (time 3h30m)
123.6. Incubate 5 minutes at RT
SECTION: 11.0 cDNA amplification and cleanup (time 3h30m)
123.7. Magnetize until clear
SECTION: 11.0 cDNA amplification and cleanup (time 3h30m)
123.8. Pull 10ul eluate and store in a new tube at -20 degrees.
SECTION: 11.0 cDNA amplification and cleanup (time 3h30m)
124. ATAC libraries are ready to sequence, cDNA requires further processing (see the next section below).  This BioA and tagmentation typically occurs on a third day.
SECTION: 12.0 cDNA tagmentation, amplification, and indexing
125. Quantify cDNA concentration using BioAnalyzer using 1ul cDNA diluted 1:10 in water (or 4.5ul water and 0.5ul DNA).  Ideally, dilute 50ng cDNA to 5ng/ul in ddH2O.  Note: expect > 50ng cDNA, if cDNA amount is low, you can use as little as 20ng cDNA for tagmentation.  Adjust the volume of DNA and H2O accordingly.

SECTION: 12.0 cDNA tagmentation, amplification, and indexing
126. Use Diagenode Tn5 and assemble transposome by mixing a 1:50 dilution (in dilution buffer) of Tn5, dilution buffer and annealed oligo, and incubate the mix at RT for 30 minutes (see recipe below).  See section 1.3 above, steps 17-21 for an overview of annealing the adapter oligos in preparation for this step.  Diluted enzyme with annealed adapter is stable for at least 6 months at -20 degrees C.

 
  
  Transposome
  
  
  Volume (ul)
  
  
  
  1x Tn5 
  
  
  1.25x(N+1)
  
  
  
  Dilution buffer
  
  
  1.25x(N+1)
  
  
  
  Annealed tagmentation
  adapter in glycerol
  
  
  2.5x(N+1)
  
  
  
  Total
  
  
  5x(N+1)
  
  

 
SECTION: 12.0 cDNA tagmentation, amplification, and indexing
127. Mix 10ul of 5ng/ul cDNA with 10ul H2O, 25ul TD buffer (recipe below), and 5ul assembled Tn5.  Incubate at 55 ºC for 5 minutes on a thermal cycler.

 
  
  2x TD buffer: 
  aliquot and store at
  -20C
  
  
  Volume (ul)
  
  
  2x Final conc
  
  
  
  H2O
  
  
  7700
  
  
   
  
  
  
  1M Tris pH 7.5
  
  
  200
  
  
  20mM
  
  
  
  1M MgCl2
  
  
  100
  
  
  10mM
  
  
  
  Dimethylformamide
  (DMF)
  
  
  2000
  
  
  20%
  
  
  
  Total
  
  
  10000
  
  
   
  
  

SECTION: 12.0 cDNA tagmentation, amplification, and indexing
128. Purify tagmented library with Qiagen column (250ul binding buffer), elute twice with 11ul EB (22ul total eluate volume
SECTION: 12.0 cDNA tagmentation, amplification, and indexing
129. Add 29ul of post tagmentation PCR mix with 1ul tube specific (sample specific) IDT8_i5_xx primer, amplify for 7 cycles.

 
  
  Post-tagmentation PCR mix
  
  
  Volume (ul)
  
  
  
  Sample in EB
  
  
  20 per sample
  
  
  
  NEBnext 2x PCR mastermix
  
  
  25x(N+1)
  
  
  
  P7 primer 25mM
  
  
  1x(N+1)
  
  
  
  H2O
  
  
  3x(N+1)
  
  
  
  Total
  
  
  50x(N+1)
  
  

 
 
  
  Tagmentation PCR
  
  
   
  
  
  
  72ºC
  
  
  5 min
  
  
   
  
  
  
  98ºC
  
  
  30 s
  
  
   
  
  
  
  98ºC
  
  
  10 s
  
  
  7 cycles
   
  ~27 min
  
  
  
  65ºC
  
  
  30 s
  
  
  
  72ºC
  
  
  60  
   
  
  

SECTION: 12.0 cDNA tagmentation, amplification, and indexing
130. After PCR reamp, add 1ul of Exonuclease I to each library and place libraries back in thermocycler for “ExoCleanup” program to get rid of excess primer.

 
  
  ExoCleanup
  
  
  
  37ºC
  
  
  15 mins
  
  
  
  80ºC
  
  
  15 mins
  
  
  
  4ºC
  
  
  Forever
  
  
  
  Total
  
  
  ~30 min
  
  

SECTION: 12.0 cDNA tagmentation, amplification, and indexing
131. Purify each sample with 35ul AMpure beads (0.7x), elute library to 50ul EB, then repeat the 0.7x purification and elute in 10ul EB.  

This double elution is to remove small MW fragments (less than ~250bp) which represent tagmentation events where the Illumina R2 is likely destroyed, and there is no actual cDNA retained (the SHARE scaffold takes up around 234bp).  Leaving these fragments present drastically reduces the efficiency of RNA sequencing, so we highly recommend you perform these steps as written!
SECTION: 12.0 cDNA tagmentation, amplification, and indexing
131.1. Bind samples and beads for 5 minutes at RT

SECTION: 12.0 cDNA tagmentation, amplification, and indexing
131.2. Magnetize for 5 minutes
SECTION: 12.0 cDNA tagmentation, amplification, and indexing
131.3. Wash 2x with fresh 80% ethanol with samples on magnet
SECTION: 12.0 cDNA tagmentation, amplification, and indexing
131.4. Dry for ~5 minutes (can place in fume hood to speed things up).  Don’t let the beads look cracked, they should look glossy.
SECTION: 12.0 cDNA tagmentation, amplification, and indexing
131.5. Resuspend in 50ul EB
SECTION: 12.0 cDNA tagmentation, amplification, and indexing
131.6. Incubate 5 minutes at RT
SECTION: 12.0 cDNA tagmentation, amplification, and indexing
131.7. Magnetize until clear
SECTION: 12.0 cDNA tagmentation, amplification, and indexing
131.8. Pull 50ul eluate and store in a new tube.
SECTION: 12.0 cDNA tagmentation, amplification, and indexing
131.9. Repeat with 35ul AMpure beads, elute in ~10ul EB and store at -20 degrees C.
SECTION: 12.0 Library quantification and sequencing
132. We recommend running all of the cDNA libraries on a bioanalyzer or tapestation to assess the average molecular weight, and the presence/absence of low MW molecules (less than 250bp), which may affect sequencing quality negatively.
SECTION: 12.0 Library quantification and sequencing
133. After confirmation of satisfactory bioanalyzer or tapestation traces, quantify all libraries to be sequenced together (in the same independently addressable lane) using a Kapa quantification kit (Roche cat number: 7960140001).  

We recommend running reactions in duplicate, using 10ul reactions, loading standards in order of ascending concentration (load NTC, then std6, std5..... std1), and using two dilutions.  We first dilute a library 1:1000 in water, then serially dilute 1:1000 again to get a 1:10^6 dilution.  We also dilute the 1:1000 again at 1:100 to get a 1:100,000 dilution.  Duplicates of both 1:10^6 and 1:100K are used to calculate the concentration of the libraries (these values will be different, that is OK).
SECTION: 12.0 Library quantification and sequencing
136. 
 

SECTION: 6.0 Hybridization
63. If you have multiple samples, put each sample in a separate BSA-coated reservoir, suitable for an 8-channel pipette (this makes loading samples across the 96 well plate easier).  Add the hybridization buffer according to the table below:

  fraction of plateul Hybridization buffer2 plates6967.281 plate3483.64½ plate1741.82⅓ plate1161.21¼ plate870.91⅛ plate435.455

 
 
SECTION: 2.0 Washing and Counting Fixed cells or nuclei (time: 30-60m)
33. Add 900ul NI-2RI (NI-RI: 1200ul NI + 3ul Enzymatic RI) to each sample, mix by inverting 
SECTION: 4.0 Optional PolyAdenylation module (time: ~45m)
50. If your samples are from hematopoietic compartment (PBMCs, HSCs), you’ll want to use the optional exogenous polyAdenylation module in this section before Reverse Transcription to improve RNA capture (since PBMCs have a lot of nucleases). If not, skip ahead to section 5.0 Reverse Transcription (starting at step 57).
SECTION: 4.0 Optional PolyAdenylation module (time: ~45m)
51. For each sample, mix 60ul cells/nuclei in NI-2RI from the previous step with 240ul PolyA master mix (recipe below), and incubate at 37ºC for 15 minutes in a thermomixer without mixing. (each 1.5mL ATAC tube is a single polyA reaction).


 

SECTION: 4.0 Optional PolyAdenylation module (time: ~45m)
52. Add 200ul NID-2RI, spin down at 1000xg, 5 minutes, RT.
SECTION: 4.0 Optional PolyAdenylation module (time: ~45m)
53. Remove supernatant and add 200ul NI-2RI, mix by inverting.
SECTION: 4.0 Optional PolyAdenylation module (time: ~45m)
54. Spin down at 1000xg, 5 minutes, RT, remove as much supernatant as possible. 
SECTION: 4.0 Optional PolyAdenylation module (time: ~45m)
55.  Resuspend so the total volume is 60ul NI-2RI.
SECTION: 4.0 Optional PolyAdenylation module (time: ~45m)
56. Proceed to step 57 of the protocol below and continue with the RT reaction.  Each polyA tube becomes a single RT reaction (7 0.2mL PCR tubes).
SECTION: 1.1 Ordering Oligo Plates and oligos for plate production
1. A visual overview of SHARE-seq:

 

SECTION: 12.0 Library quantification and sequencing
137. 
SECTION: 12.0 Library quantification and sequencing
135.     
The Epstein lab typically sequences libraries on the NovaSeq X 10B, as it has the
lowest cost per base.  The sequencing read configuration is as below, for separated(!!) cDNA and ATAC-seq libraries.  We find separating the libraries allows for better quality sequencing data, likely due to the differences in molecularweights between cDNA and ATAC.
SECTION: 12.0 Library quantification and sequencing
134. Quantify using the Kapa Quant qPCR kit for Illumina, and pool libraries to 4-10nM concentration.  Expect > 30nM, average library size of 400bp with minimal adapter dimers.