Supplementary Table. PAIR Protocol Check List

Material and Reagents – Day 1

/
Per sample
/
Experiment ( x )
Pharmacological Drugs e.g. DHPG: 10mM->20M;
PNA: 5M->50nM;
NB+B27:
HEPES Buffered Saline:
TX-100 Lysis Buffer:
  • Protease Inhibitor
/ 8l / 4ml
40l /4ml
4-8ml (± Drug Rx)
8ml
4ml
  • ____l / __l
/ 




  • ____l / __l

Procedure-Day 1

/ Steps /
Time/Date
/

Notes

PNA in cells / Check Cell Healthiness
DHPG/NB+B27 Treatment8l / 4 ml
PNA/NB+B27 40l / 4 ml / 30 min 16ul + 4 ml
90 min
UV-crosslinking / Rinse with HEPES BS (Cold)4 ml
Replace with HEPES BS (Cold)4 ml
UV Crosslink / 

2.5 min
Collecting cells / Replace with Lysis buffer4 ml
Scrape cell and transfer to 15ml tube
Save TCL for gel analysis30 l / 


Cells Lysis / Store Samples in –80oC O/N or at least 1 hr / 

Material and Reagents – Day 2

/
Per sample (from 4ml)
/
Experiment ( x )
TX-100 Lysis Buffer:
  • Protease Inhibitor
Salt Free Lysis Buffer
  • Protease Inhibitor
Rnase A1 g/l->1 g/ml / 2ml
  • ____l / __l
100 l
  • ____l / __l
4 l/4ml / 
  • ____l / __l

  • ____l / __l

Procedure-Day 2

/ Steps /
Time
/ Notes
RNAse treatment / Thaw tubes in cold H2O
RNAse A 4l/4ml
Rotate, 37oC / 

20 min
Coupling of PNA to the beads / Add Beads 10l/4ml
Rotate, 37oC
Rotate, RT
Centrifuge, 20K
Save FT for gel analysis30 l / 
30 min
30 min
1 min

Wash / Transfer beads to 1.5ml tube
Magnetic Stand
Aspirate; Add Lysis buffer500-1000 l
Rotate, RT
Magnetic Stand
Aspirate; Add Lysis buffer500-1000 l
Rotate, RT
Magnetic Stand
Aspirate / 
10 min

20 min
10 min

20 min
10 min

Elution / Add Salt-free Lysis buffer100 l
Incubate 50oC in thermomixer
Spin in desktop centrifuge
Magnetic separate
Transfer eluate to new tube100 l / 
20 min
1 min
5-10 min

Table 1. PAIR Protocol Check List (cont’d)

Material and Reagents – Day 2/3

/
Per sample
/
Experiment ( x )
Methanol
Chloroform
Water
NH4HCO3 100 mM->25 mM / 700 l
100 l
300 l
50 l / 



Procedure-Day 2/3

/ Steps /
Time
/ Notes
Chloroform-Methanol Precipitation / Add methanol400 l
Vortex and spin, 10K rpm
Add chloroform100 l
Vortex and spin, 10K rpm
Add H2O300 l
Vortex and spin, 10K rpm 4oC
Aspirate and d/c top phase
Add methanol to lower phase300 l
Vortex and spin, 13K rpm 4oC
Discard methanol; Air dry
Resuspend pellet in NH4HCO350 l
Save 20l for gel analysis / 10 sec

10 sec

 3 min


10 min
5~16min


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