Alice:LabNotes/2009-9-15

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Single-end solexa sequencing library[edit]

Materials[edit]

use previously captured samples
capture reaction setup can be found under LabNotes 8-15-09 and 8-16-09
samples to be used: D3(6&7 subsets of NA12878), DF6-9-9, foreskin, ips(PGP1-ips), Cvi(CV-ips), CVf(CV-fibroblast)
Check LabNotes 9-14-09 for the details on re-amplification PCR and the ratio to pool all the products together

Procedure[edit]

Reagent setup:

(Pair-end sequencing library construction)
Solexa_1 adaptor and Solexa_2_PE adaptor will be used
for PCR at the last step use these primers:  Solexa_PCR_PE_loH & Solexa_PCR_upH

(single-end sequencing library construction)
Solexa_1 adaptor and Solexa_2 adaptor will be used
for PCR at the last step use these primers:  Solexa_PCR_up Solexa_PCR_lo

PCR re-amplification[edit]

 AmpF6.3/AmpR6.3 and dUTP:dNTP 1:40 

 system setup:                                                   x4          
 H2O                                                  46ul      184ul  
 2x KAPA Master mix                                   50ul      200ul     
 dUTP(1mM)                                             4ul       16ul      
 AmpF6.3NH2(100uM)                                   0.4ul        8ul      
 AmpR6.3NH2(100uM)                                   0.4ul        8ul     
 template                                            0.5ul        2ul     
 
  95C 30sec -> (95C 3sec -> 60C 30sec) x 3 -> 60C 2min -> 15C hold.
Qiaquick column purification and elute in 30ul EB.
Nanodrop:
D3: 19.2ng/ul         DF: 33.3ng/ul          fs: 17.7ng/ul
ips: 13ng/ul          cvi: 23.7ng/ul         cvf: 12.2ng/ul

Digestion with MmeI[edit]

 (dUTP 250uM,167.3ng/ul of product will use 6ul of DNA along with 2ul of 1mM SAM and 8ul of 2U/ul MmeI) 
                                DF & cvi       ips & cvf       D3 & fs 
 DNA                              30ul          30ul           30ul
 10X NEBuffer 4                   5ul           4ul            5ul
 1mM SAM(fresh)                   6ul           3ul            4ul
 2U/ul Mme I                      8ul           3ul            5ul
 ddH2O                            1ul            -             6ul

 1mM SAM: 32mM SAM 1ul + 31ul ddH2O.
 37C 2h
MinElute column purify. Elute in 11ul EB.

USER digestion[edit]

 DNA                    10ul         
 USER                    3ul     
 total                  13ul           
 37C 4hr

S1 nuclease digestion[edit]

 10 x S1 nuclease buffer:   2ul     
 DNA after USER digestion: 13ul    
 S1 nuclease (10U/ul):      1ul     
 ddH2O                      4ul     
 37C 10mins.
 Minelute cloumn purify. Elute in 16ul H2O.

end repair[edit]

 Total                  25ul            
 DNA                    15ul           
 dNTP                    5ul          
 10xendrepair buffer   2.5ul          
 enzyme                  1ul          
 ATP(10mM)               5ul        
                                  
 
Keep at room temperature (~25C) for 45 minutes. Purify with Minelute. Elute in 20ul H2O.
PAGE size selection
File:ZhangLab 2 2009-09-16 12hr 47min.jpg   File:ZhangLab 2 2009-09-16 12hr 48min.jpg

adapter ligation[edit]

adaptor construction: add 10ul Solexa_2_PE_up and 10ul Solexa_2_PE_lo_noP. 
                          10ul Solexa_2_upNH2 and 10ul Solexa_2_lo_noP. 
                          10ul Solexa_1_upNH2 and 10ul Solexa_1_lo_noP.             
95C 5mins -> 65C 30mins -> 4C.


total                           30ul       
 DNA                            13ul             
 100uM Solexa_1 adaptor          1ul        
 100uM Solexa_2 adaptor          1ul         
 2xQuickLiage buffer            15ul          
 QuickLigase enzyme(NEB)         1ul           
 extra ATP(10mM)               2.5ul        


 Keep at room temperature (~25C) for 10 minutes. Purify with Minelute column. Elute in 10ul H2O.

Nick-translation[edit]

 DNA                         10ul               
 10x ThermoPol buffer       1.2ul          
 10mM dNTP                  0.5ul                  
 Bst polymerase(8U/ul)      0.5ul             
 65C for 12 minutes

PCR of sequencig library[edit]

 Nick-translated DNA               6ul             
 Solexa_PCR_up(100uM)            0.2ul             
 Solexa_PCR_lo(100uM)            0.2ul            
 2xiProof master mix              50ul           
 50x SYBG                        0.2ul          
 ddH2O                            44ul        
 
 98C 30sec -> 7-8 cycles of (98C 10sec -> 65C 20 sec -> 72C 20sec) -> 72C 3min ->15C hold.
 PAGE size selection 175-225bp
 File:ZhangLab 2 2009-09-17 16hr 21min-1.jpg

QPCR[edit]

template                  1ul      
Phusion 2x master mix     25ul
Syb FP5(100uM)            0.2ul
Syb RP7(100uM)            0.2ul
50X SYBR green            0.2ul
H2O                       24ul
98C 30sec -> (98C 10sec -> 58C 20sec -> 72C 20 sec)X21 -> 72C 3min -> 15C
results:
DF6-9-9:         12.3nM
foreskin:        5nM
CV-ips:          26.3nM
CV-fibroblast:   10nM