G eenlea Lab SHARE-seq P o ocol
1 o 15
Supplemen a y No e 1: SHARE-seq p o ocol
Gene al no es
● Be o e s a ing he expe imen , clean ma e ials wi h RNase Away and 70% e hanol, use au ocla ed ips and
ubes. Main ain samples and solu ions ice-cold du ing he whole expe imen i no speci ied o he wise.
● De aul spin o cells a 500xg o 5 min w/ swing bucke cen i uge.
● S a wi h 100k-1M cells o ixa ion (mo e he be e bu ensu e enough combina o ial space o inal sublib a ies)
o Expec ~20% o o iginal s a ing numbe o cells by lib a y p ep s age.
● Resuspend oligos o app op ia e concen a ions in IDTE bu e :
o 1mM = linke oligos
o 100µM = RT p ime , TSO, ba coded oligos
● Add all RNase inhibi o s esh o mas e mix.
● Enzyma ics RI is he only RNase Inhibi o ha seems o be compa ible wi h Tn5 ac i i y, so we swi ch cells om
he PBS-2RI in o PBS-RI bu e be o e ATAC.
● Some washing s eps speci ies keeping pelle /beads s able and adding bu e di ec ly wi hou dis u bing he
pelle /beads. This does a ec inal eco e y.
● When emo ing supe na an , use a wo-s ep emo al me hod: i s emo e he majo i y o supe na an wi h
P1000, and hen emo e he emaining amoun wi h P200. I may help o il he 2mL ube comple ely ho izon ally
as you ge close o emo ing all he liquid o a oid emo ing he pelle while emo ing as much liquid as possible.
● 2mL lo-bind Eppendo ubes has be e cell/nuclei eco e y han 1.5mL ubes only when cen i uga ion is
pe o med in a swing bucke because i ’s easie o comple ely aspi a e he supe na an om 2mL ubes wi hou
dis u bing he pelle a he ip o he 2mL ube.
● We spli he bulk ATAC and RT eac ion pe sample in o smalle ubes because he eac ion e iciency is be e
wi h a smalle olume (e en unde he same concen a ion o eac ion componen s).
● Whe e possible, check cell/nuclei quali y and coun using Coun ess a e e y majo s eps (ideally: p e- ix, p e-
ATAC, p e-spli pool, pos -liga ion. I necessa y, can ge away wi hou coun ing a p e- ix).
● Check be o e e e y un o ensu e su icien eagen s in s ock.
G eenlea Lab SHARE-seq P o ocol
2 o 15
S ock Bu e ecipes:
These bu e s can be made in big ba ch ahead o ime and s o ed o a ew mon hs
Nuclei Isola ion Bu e (NIB) (Keep a 4˚C)
S ock solu ion
Final conc.
Vol o add (mL)
1M T is HCl pH 7.5
10mM
0.5
5M NaCl
10mM
0.1
1M MgCl2
3mM
0.15
10% NP40 (IGEPAL CA-630)
0.1%
0.5
Ul apu e wa e
48.75
To al
50
2x RCB (Keep a oom emp)
S ock solu ion
Final conc.
Vol o add (mL)
1M T is HCl pH 8.0
100mM
1
5M NaCl
100mM
0.2
20% SDS
0.4%
0.2
Ul apu e wa e
8.58
To al
10
2x BW (Keep a 4˚C)
S ock solu ion
Final conc.
Vol o add (mL)
1M T is HCl pH 8.0
10mM
0.5
5M NaCl
2M
20
0.5M EDTA
1mM
0.1
Ul apu e wa e
29.4
To al
50
1x B&W-T (Keep a 4˚C)
S ock solu ion
Final conc.
Vol o add (mL)
1M T is HCl pH 8.0
5mM
0.25
5M NaCl
1M
10
0.5M EDTA
0.4%
0.05
10% Tween 20
0.05%
0.25
Ul apu e wa e
39.45
To al
50
IDTE (oligo esuspension) (Keep a oom emp)
S ock solu ion
Final conc.
Vol o add (mL)
1M T is HCl pH 8.0
10mM
0.5
0.5M EDTA
0.1mM
0.01
Ul apu e wa e
49.5
To al
50
G eenlea Lab SHARE-seq P o ocol
3 o 15
STE (Keep a oom emp)
S ock solu ion
Final conc.
Vol o add (mL)
1M T is HCl pH 8.0
10mM
0.5
5M NaCl
50mM
0.5
0.5M EDTA
1mM
0.1
Ul apu e wa e
48.9
To al
50
2x TD bu e (Keep a -20C)
S ock solu ion
Final conc.
Vol o add (mL)
1 M T is-HCl pH 7.6
20 mM
2 mL
1 M MgCl2
10 mM
1 mL
*I p epa ing T is om solid base, adjus pH o 7.6 be o e he
addi ion o DMF
Dime hyl Fo mamide (DMF)
20%
20 mL
Ul apu e wa e
NA
B ing up o 100
mL
To al
100 mL
G eenlea Lab SHARE-seq P o ocol
4 o 15
Spli pool oligo pla e p epa a ion:
Can be made in big ba ch ahead o ime and s o ed a -20C o a ew mon hs
1. Thaw Round1, 2, 3 ba code oligo pla es a oom emp and le comple ely haw ( ew hou s o also y wa e ba h).
a. Ve y c i ical s ep ha all oligos a e a oom emp be o e use.
b. Spin he pla es down using a pla e adap e on a swing bucke o pla e-compa ible cen i uge.
2. P epa e each linke oligo dilu ion in ubes as below:
a. 120µL Round 1 linke oligo (1mM) + 11,880µL STE bu e
b. 120µL Round 2 linke oligo (1mM) + 9,480µL STE bu e
c. 144µL Round 3 linke oligo (1mM) + 9,360µL STE bu e
3. Dispense ollowing amoun s o linke oligos in o each 96 well PCR pla e:
a. 90µL dilu ed Round 1 linke oligo
b. 88µL dilu ed Round 2 linke oligo
c. 86µL dilu ed Round 3 linke oligo
4. Use a mul ichannel pipe e o ans e he ba code oligo pla es o he app op ia e Round linke pla e:
a. 10µL Round 1 ba code oligos
b. 12µL Round 2 ba code oligos
c. 14µL Round 3 ba code oligos
5. Seal pla es wi h aluminum adhesi e co e and ensu e ha each well is well p o ec ed.
Se up he mocycle as below:
Temp
Time
95˚C
2:00 min
-1˚C/1 min
20˚C
2:00 min
4˚C
Hold
To al
~1h 26min
6. Check each well o make su e ha he e was no signi ican e apo a ion. I so, add app op ia e amoun o wa e o
compensa e.
7. Aliquo 10µL o he annealed oligos o a new pla e (should be enough o 9x pla es o each ound). Use a
liquida o o a liquid handling pla o m o dis ibu ion. Seal ca e ully o ensu e p ope seal a each well wi h
aluminum oil seal and s o e a -20˚C.
G eenlea Lab SHARE-seq P o ocol
5 o 15
Bu e p epa a ion
P epa e ollowing bu e s esh on he day o expe imen , keep on ice unless o he wise speci ied.
Recommended o de o p epa e bu e s: PBS-2RI, 2x TB Omni, 1x TB Omni, PBS-RI, NIB-RI.
Scale acco ding o he numbe o samples and numbe o eac ions needed
PBS-2RI
Vol o 1
sample (µL)
1xPBS
3000
7.5% BSA
16.05
Enzyma ics RI
7.5
SUPERase RI
3.75
To al
3027.3
PBS-RI
Vol o 1 xn
(µL)
1x PBS
5
Enzyma ics RI
0.0125
To al
5.0125
NIB-RI
Vol o 1
sample (µL)
NIB
8323
Enzyma ics RI
21
SUPERase RI
21
To al
8365
*6.565mL ixed olume o NIB-RI equi ed gi en
ound 1 ba coding uses a single 96 well pla e,
ano he 1.8mL pe sample equi ed
2x TB (Omni)
Vol o 1 xn
(µL)
0.2M T is-ace a e
8.25
5M K-ace a e
0.66
1M Mg-ace a e
0.5
10% Tween-20
0.5
1% Digi onin
0.5
100% DMF
8
Ul apu e wa e
6.59
To al
25
G eenlea Lab SHARE-seq P o ocol
6
ATAC (~20min)
* Includes ime-sensi i e s eps, make su e he app op ia e bu e s a e eady ahead o ime.
* This p o ocol assumes nuclei isola ion, pe meabiliza ion, and ixa ion has al eady occu ed du ing issue dissocia ion.
1. Se swing bucke cen i uge o 4C.
2. P epa e 1xTB:
1x TB Omni
Vol o 1 xn
(µL)
2x TB (Omni)
25
Ul apu e wa e
16.45
PIC
0.2
Enzyma ics RI
0.85
To al
42.5
3. A e app op ia e nuclei dissocia ion p o ocol and ixa ion, mix he desi ed amoun o cells wi h 500µL o PBS-RI
and mix ho oughly, especially i i con ains iodixanol.
4. Spin down a 500xg o 5 min a 4C in swing bucke cen i uge.
5. P epa e bulk ansposi ion as ollows, each eac ion is ~40k cells. P epa e jus enough based on he o al numbe
o ATAC eac ions needed.
ATAC xn
Vol o 1 xn
(µL)
Sample esuspended
in PBS-RI
5
1xTB (Omni)
42.5
SHARE-ATAC Tn5
WJG Ma 2023
2.5
To al
50
a. Resuspend each sample pelle in PBS-RI in app op ia e olumes (5 µL pe ATAC eac ion).
b. Assemble he ATAC eac ions pe sample wi h app op ia e olume o 1xTB and Tn5. Tn5 is iscous and
should be pipe e mixed in slowly.
c. Mix well and dis ibu e 50µL pe well o a lo-bind 96 well pla e on a cooling block on ice (i ’s ok i he las
well ge s <50µL). Keeping pla e cold is essen ial o con ol he Tn5 ac i i y be o e he eac ion s a s. Seal
pla e, and incuba e a 37˚C o 30 min wi h shaking a 500 pm.
d. A his ime, haw he 5x RT bu e , RT p ime (100µM), and dNTPs.
6. Pool ATAC eac ions by sample in 2mL lo-bind ubes, keeping he pla e and ubes on ice as much as possible.
7. Spin down a 500xg o 5 min a 4C, emo e supe na an .
8. Wash pelle gen ly wi hou esuspending wi h 1mL NIB-RI.
9. Spin down a 500xg o 5 min a 4C.
10. Remo e supe na an and esuspend pelle ho oughly in app op ia e olumes o EB pe sample (10µL pe RNA
RT eac ion).
G eenlea Lab SHARE-seq P o ocol
7
RT (~45 min)
1. P epa e RT mix (op imized o 100k cells pe 50µL xn):
RT mix
Vol o 1 xn (µL)
5x RT bu e
10
Ul apu e wa e
1.56
dNTPs
2.5
Enzyma ics RI
0.31
SUPERase RI
0.63
RT p ime (100µM)
5
50% PEG (wide bo e ips)
15
Maxima H Minus RT (add
igh be o e xn)
5
To al
40
2. S a he RT p o ocol on he mocycle – he p o ocol should ha e a 50˚C hold so ha when he samples a e pu in
he machine, he hold is eleased and he 10min a 50˚C can be s a ed igh away.
3. Add app op ia e olumes o RT mix o cells in EB (40uL RT mix + 10uL sample pe eac ion).
4. Spli RT mix in o PCR s ips wi h 50µL/ ube.
5. Se up he mocycle as below:
Temp
Time
50˚C
Hold
50˚C
10 min
8˚C
12s
3 cycles
15˚C
45s
20˚C
45s
30˚C
30s
42˚C
2 min
50˚C
3 min
50˚C
5 min
To al
37 min
6. All s eps below a e a oom empe a u e.
7. Thaw ba code oligos pla es, block 1, 2, 3 oligos and T4 DNA ligase bu e a oom emp. Place NIB-RI a oom
emp. Se cen i uge o oom emp.
8. Add 300µL NIB-RI o a new 2ml lobind ube pe sample and pool RT eac ions in o i , pipe e mix well.
9. Spin down a 500xg o 5 min a oom emp in swing bucke cen i uge.
10. Wash wi h 500µL NIB-RI wi hou dis u bing he pelle .
11. Spin down a 500xg o 5 min a oom emp in swing bucke cen i uge.
12. Remo e supe na an and esuspend pelle ho oughly wi h app op ia e olumes o NIB-RI. The o al olume o a
single 96 well pla e o hyb idiza ion is 1080µL NIB-RI. Di ide by he numbe o independen samples.
a. Coun nuclei o con i m nuclei quali y and cell coun (p e-spli pool)
G eenlea Lab SHARE-seq P o ocol
8
Hyb idiza ion-liga ion (~5h s)
1. Make su e ha all oligo pla es a e hawed o oom emp and spun down be o e hyb idiza ion.
2. P epa e hyb idiza ion bu e in 5mL ube:
Hyb idiza ion bu e
Vol o 96 wells
(µL)
Ul apu e wa e
2589.3
T4 ligase bu e (10x)
540
SUPERase RI
13.5
Enzyma ics RI
43.2
10% NP-40
54
To al
3240
3. Add app op ia e olumes o hyb idiza ion bu e o esuspended samples in NIB-RI, mix well. I he e a e mul iple
samples, hen di ide he hyb idiza ion bu e app op ia ely o numbe o sample.
4. Using a mul ichannel ough, aliquo 40µL sample mix u e o Round 1 pla e, mix, and seal pla e.
5. Shake a 300 pm o 30min a 25oC.
6. P epa e blocking oligo 1 mix u e:
Blocking oligo 1
Vol (µL)
Round 1 blocking (1mM)
25.3
T4 ligase bu e (10x)
211.2
Ul apu e wa e
915.5
To al
1152
a. Dis ibu e 90µL each o a PCR s ip (12x) and use a P20 mul ichannel o dis ibu e blocking oligo and mix.
7. Using a whole new P10 pipe e ip box, add 10µL blocking oligo 1 mix u e o each well, mix, and seal pla e.
8. Shake a 300 pm o 30 min a 25oC.
9. Pool samples in o a ough, and using a whole new P200 pipe e ip box, aliquo 50µL o mix u e o Round 2 pla e.
a. Use a P200 mul ichannel o aspi a e and pool in o a new basin and edis ibu e using a mul ichannel.
10. Mix, seal pla e, and shake a 300 pm o 30min a 25oC.
11. P epa e blocking oligo 2 mix u e:
Blocking oligo 2
Vol (µL)
Round 2 blocking (1mM)
30.4
T4 ligase bu e (10x)
211.2
Ul apu e wa e
910.4
To al
1152
a. Dis ibu e 90µL each o a PCR s ip (12x) and use a P20 mul ichannel o dis ibu e blocking oligo and mix.
12. Using a whole new P10 pipe e ip box, add 10µL blocking oligo 2 mix u e o each well, mix, and seal pla e.
13. Shake a 300 pm o 30min a 25oC.
14. Pool samples in o a ough, using a whole new P200 pipe e ip box, aliquo 60µL o mix u e o Round 3 pla e.
a. Use a P200 mul ichannel o aspi a e and pool in o a new basin and edis ibu e using a mul ichannel.
15. Mix, seal pla e, and shake a 300 pm o 30min a 25oC.
16. P epa e blocking oligo 3 mix u e:
Blocking oligo 3
Vol (µL)
Round 3 blocking (1mM)
26.5
10% NP-40 (can be
eplaced wi h wa e )
11.5
Ul apu e wa e
1114
To al
1152
b. Dis ibu e 90µL each o a PCR s ip (12x) and use a P20 mul ichannel o dis ibu e blocking oligo and mix.
17. Using a whole new P10 pipe e ip box, add 10µL blocking oligo 3 mix u e o each well. mix, and seal pla e.
18. Shake a 300 pm o 30min a 25oC.
19. Pool samples o a ough and add 70µL 7.5% BSA o he solu ion and mix well.
c. Can il e while ans e ing using a FlowMi 40µm il e o emo e mul iple s.
G eenlea Lab SHARE-seq P o ocol
9
20. T ans e o 4x lo-bind 2mL ubes, spin down a 500xg o 5 min a oom emp in a swing bucke cen i uge.
21. Remo e supe na an and add gen ly 1mL NIB-RI o pelle wi hou esuspending.
22. Spin down a 500xg o 5 min a oom emp, emo e supe na an .
23. Resuspend wi h 85µL NIB-RI: s a wi h 40µL, pool all ubes, measu e olume and op up o 85 µL.
24. P epa e Liga ion mix:
Liga ion bu e
Vol (µL)
Ul apu e wa e
251.8
T4 ligase bu e (10x)
40
SUPERase RI
1
Enzyma ics RI
3.2
10% NP-40
4
T4 ligase
20
To al
320
25. Mix 80µL sample o 320µL liga ion mix.
26. Dis ibu e 50µL o 8 PCR ubes.
27. Shake a 300 pm o 30 min a 25oC.
28. Pool samples, spin down a 500xg o 5 min a oom emp.
29. Remo e supe na an and wash pelle wi h 1mL NIB-RI.
30. Spin down a 500xg o 5 min a oom emp.
31. Resuspend ho oughly (10x pipe ing) wi h **200µL NIB-RI and coun nuclei (pos -liga ion).
d. Volume o inal esuspension depends on expec ed nuclei eco e y. Gene ally, can assume be ween
100-200k bu will change depending on cell ype and he numbe o cells a he s a o spli and pool.
e. **400µL assumes 200k inal eco e y. Adjus app op ia ely o desi ed numbe o cells pe lib a y.
Re e se c osslinking & pull down (2:45 h s)
1. Based on desi ed sub-lib a y size (# cells), dilu e he cells app op ia ely o each 50µL eac ion.
a. Range: 2,000-20,000 cells / 50µL.
b. Each sub-lib a y is p ocessed independen ly.
c. N = numbe o sublib a ies
2. P epa e e e se c osslinking mas e mix as ollows.
RC MM
Vol o 1 sublib
(µL)
2x RCB Bu e
50
p o einase K (20mg/mL)
2
SUPERase RI
1
To al
53
3. Fo each ube o 50µL sample, add 53 µL o RC MM.
4. Incuba e a 55˚C o 1h.
a. Do no incuba e longe , dec eases yield
5. Recommended s opping poin , s o e p oduc a -80˚C o ew weeks
6. Thaw lib a ies, i necessa y, PMSF/IPA, equilib a e B&W bu e s o oom emp.
7. A e haw, s op p o einase K by adding 5µL 100mM PMSF/IPA (don’ eeze haw) and incuba e a oom emp o
10 min.
8. P epa e ollowing bu e s (N = numbe o sublib a ies):
1x B&W-T/RI
Vol o 1 sublib (µL)
1x B&W-T
400
SUPERase RI
4
To al
404
