Noi/NOTES/2012-8-1: Difference between revisions

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* Continued from: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2012-7-27
* Continued from: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2012-7-27
* I have sheared gDNA from the previous batch left ~22ul (conc. ~40ng/ul based on conc. from the HAPMAP plate). I added 25ul H2O to each tube -> total volume 47ul, and will prepare end repair reaction mix in 23ul
* I have sheared gDNA from the previous batch left ~22ul (conc. ~40ng/ul based on conc. from the HAPMAP plate). I added 25ul H2O to each tube -> total volume 47ul, and will prepare end repair reaction mix in 23ul
* Note: Since I start with sheared 880ng, I reduce the amount of enzymes in each step, but keep total volume the same
'''End repair''' 
Total reaction in            70ul
DNA                       47ul (amount ~880ng)
End repair reaction mix      23ul
KAPA End repair mix 1x rxn 8x rxn mix
10X End repai buffer 7.00 56.00
KAPA End repair enzyme 3.00 24.00
H2O               13.00 104.00
==================================================
Total               23.00 184.00 
Aliquot to column #1: 23ul
'''A-tailing'''
Total reaction in 30ul
A-tailing reaction mix       1x rxn 8x rxn mix
KAPA 10X A-tailing buffer      3.00 24.00
KAPA A-tailing enzyme          2.00 16.00
H2O                       25.00 200.00
==================================================
Total                       30.00 240.00
Aliquot to column #2: 30ul
'''Adapter ligation'''
Total reaction in 40ul
Ligation reaction mix 30ul
Methylation adapter  10ul
Ligation reaction mix         1x rxn 8x rxn mix
KAPA 5X Ligation buffer        8.00 64.00
KAPA DNA ligase                 3.00 24.00
H2O                       19.00 152.00
Total                       30.00 240.00
'''Methylation adapter preparation'''
''Alan's note:''
1. Use the methylated adapter with paired-end pcr primer pairs(PCR primer PE1.0 and PCR primer PE 2.0) provided in illumina's paired end genomic DNA sample prep kit
2. Use 10ul of methylated adapter to 5ug gDNA insert, if starting DNA amount is less than 5ug, titrate the volume of adapter reagent accordingly to maintain 10:1 ratio of DNA
Since I used 1ug of DNA, used 2ul of adapters, but dilute to final volume 10ul (This volume is larger than KAPA protocol, but the total amount is the same)
Methylation adapter 2ulx 8rxn 16.00
H2O                 8ulx8rxn 64.00
==================================================
Total                         80.00
Aliquot to column #3: 30ul
20C for 15min
'''Reagent preparation'''
'''Reagents'''                     '''1rxn''' '''Min volume in 30mL reservior'''
AMPure beads                     120ul ~1.1mL
20% PEG/2.5M NaCl     90+50 = 140ul ~1.4mL
80% EtOH                    140x6 = 840ul      ~10.0mL
H2O for elution                         23ul ~1.0mL
==epMotion run ==
'''Report file of epMotion run:'''
== Amplification of bisulfite sequencing llibraries with N2 barcoded primers ==
== Amplification of bisulfite sequencing llibraries with N2 barcoded primers ==
* Test if the adapter ligation reaction worked before amplify the libraries with barcoded primers
* Test if the adapter ligation reaction worked before amplify the libraries with barcoded primers
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* Aliquot 88ul, add 2ul of 10uM PCR_R Ind (Ind1-8), 10ul of bis-cvt DNA template
* Aliquot 88ul, add 2ul of 10uM PCR_R Ind (Ind1-8), 10ul of bis-cvt DNA template
  '''Program'''<br>
  '''Program'''<br>
  98C 2min (98C 15sec -> 64C 20sec -> 72C 30sec) x10cycles -> 72C 3min -> 15CHold
  98C 2min (98C 15sec -> 64C 20sec -> 72C 30sec) x10cycles -> 72C 3min -> 15CHold
{| {{table}}  
{| {{table}}  
| width="80pt" align="center" style="background:#f0f0f0;"|'''Sample ID'''
| width="80pt" align="center" style="background:#f0f0f0;"|'''Sample ID'''

Revision as of 23:38, 3 August 2012

[Link to calendar]

WGBS sequencing library preparation using KAPA protocol (Eppendorf) and epMotion (Repeat!)

  • Continued from: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2012-7-27
  • I have sheared gDNA from the previous batch left ~22ul (conc. ~40ng/ul based on conc. from the HAPMAP plate). I added 25ul H2O to each tube -> total volume 47ul, and will prepare end repair reaction mix in 23ul
  • Note: Since I start with sheared 880ng, I reduce the amount of enzymes in each step, but keep total volume the same
End repair  
Total reaction in             70ul	
DNA 	                       47ul (amount ~880ng)
End repair reaction mix       23ul

KAPA End repair mix	1x rxn	8x rxn mix
10X End repai buffer	7.00	56.00
KAPA End repair enzyme	3.00	24.00
H2O	               13.00	104.00
==================================================
Total	               23.00	184.00  
Aliquot to column #1: 23ul
A-tailing
Total reaction in 30ul

A-tailing reaction mix	      1x rxn	8x rxn mix
KAPA 10X A-tailing buffer      3.00	24.00
KAPA A-tailing enzyme  	        2.00	16.00
H2O	                       25.00	200.00
==================================================
Total	                       30.00	240.00
Aliquot to column #2: 30ul
Adapter ligation
Total reaction in 40ul 
Ligation reaction mix 30ul
Methylation adapter   10ul

Ligation reaction mix	         1x rxn 	8x rxn mix
KAPA 5X Ligation buffer         8.00	64.00
KAPA DNA ligase	                3.00	24.00
H2O	                       19.00	152.00
Total	                       30.00	240.00

Methylation adapter preparation
Alan's note: 
1. Use the methylated adapter with paired-end pcr primer pairs(PCR primer PE1.0 and PCR primer PE 2.0) provided in illumina's paired end genomic DNA sample prep kit
2. Use 10ul of methylated adapter to 5ug gDNA insert, if starting DNA amount is less than 5ug, titrate the volume of adapter reagent accordingly to maintain 10:1 ratio of DNA
Since I used 1ug of DNA, used 2ul of adapters, but dilute to final volume 10ul (This volume is larger than KAPA protocol, but the total amount is the same)

Methylation adapter	2ulx 8rxn	16.00
H2O	                 8ulx8rxn	64.00
==================================================
Total		                        80.00
Aliquot to column #3: 30ul
20C for 15min
Reagent preparation
Reagents 	                     1rxn	Min volume in 30mL reservior
AMPure beads	                    120ul	~1.1mL
20% PEG/2.5M NaCl	    90+50 = 140ul	~1.4mL
80% EtOH        	            140x6 = 840ul      ~10.0mL
H2O for elution	                        23ul 	~1.0mL

epMotion run

Report file of epMotion run:

Amplification of bisulfite sequencing llibraries with N2 barcoded primers

  • Test if the adapter ligation reaction worked before amplify the libraries with barcoded primers
  • Loaded 2.5 ul of bis-cvt DNA and 2.5ul of amplified amplicons

File:ZhangLab 2 2012-08-02 11hr 31min-HAPMAP-bis-test.jpg

Components 1 rxn 26rxn mix
DNA template 10.00 0.00
10uM PCR_F 2.00 52.00
10uM PCR_R.Indx (1-8) 2.00 0.00
2x iQ Supermix 50.00 1,300.00
H2O 36.00 936.00
  • Aliquot 88ul, add 2ul of 10uM PCR_R Ind (Ind1-8), 10ul of bis-cvt DNA template
Program
98C 2min (98C 15sec -> 64C 20sec -> 72C 30sec) x10cycles -> 72C 3min -> 15CHold
Sample ID Labeling Indx
WGBS.GM12891 #1 PCR_R.N2Indx1
WGBS.GM12892 #2 PCR_R.N2Indx2
WGBS.GM10861 #3 PCR_R.N2Indx3
WGBS.GM11994 #4 PCR_R.N2Indx4
WGBS.GM11995 #5 PCR_R.N2Indx5
WGBS.GM18506 #6 PCR_R.N2Indx6
WGBS.GM18507 #7 PCR_R.N2Indx7
WGBS.GM18505 #8 PCR_R.N2Indx8
  • Purified sequencing libraries with 0.8x vol AMPure bead with epMotion (3 strips), eluted with 25ul EB for each well -> total volume at the end ~70ul

File:ZhangLab 2 2012-08-02 16hr 05min bead-HAPMAP-WGBS.jpg

PAGE quantification of 400-600bp fragments

  • Dr. Zhang suggested to cut the band around 400-600bp because 250bp is too short for PE sequencing.
  • Perform PAGE size-selection of the size range 400-600bp for a small scale sequencing. Then pooled 60ng of each library and performed PAGE size-selection.

File:ZhangLab 2 2012-08-02 16hr 05min bead-HAPMAP-WGBS-400-600bp PQ.jpg

Lab IDs Final conc.

of 400-600bp (ng/ul)

Total yields in 70ul (ng) Volume for 60ng (ul)
#1 13.50 945.28 4.44
#2 9.66 675.94 6.21
#3 11.24 786.94 5.34
#4 13.64 955.09 4.40
#5 13.85 969.55 4.33
#6 8.95 626.85 6.70
#7 12.40 867.78 4.84
#8 11.29 790.44 5.31


PAGE size-selection

File:ZhangLab 2 2012-08-02 18hr 23min-PAGE-SS.jpg

  • Note: From the image, it seemed like I cut the gel a little smaller than 600bp
  • Resuspend with 12ul H2O each tube -> total volume 24ul.
  • Quantify concentration by PAGE quantification (also verify the size of sequencing libraries) and by dsDNA Qubit
  • Library ID: NP_WGBS_HAPMAP1-8_July27