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
* Note: Since I start with sheared 880ng, I reduce the amount of enzymes in each step, but keep total volume the same (except ligation 50ul -> 40ul total)
== End repair ==
'''End repair'''
{| {{table}}
Total reaction in            70ul
| width ="150pt" align="center" style="background:#f0f0f0;"|'''Total reaction'''
DNA                       47ul (amount ~880ng)
| align="center" style="background:#f0f0f0;"|'''70.00'''
End repair reaction mix       23ul
| align="center" style="background:#f0f0f0;"|'''ul'''
|-
KAPA End repair mix 1x rxn 8x rxn mix
| DNA ||47.00||ul
10X End repai buffer 7.00 56.00
|-
KAPA End repair enzyme 3.00 24.00
| End repair reaction mix ||23.00||ul
H2O               13.00 104.00
|}<br>
==================================================
{| {{table}}
Total               23.00 184.00
| width ="150pt" align="center" style="background:#f0f0f0;"|'''KAPA End repair mix'''
Aliquot to column #1: 23ul
| width ="80pt" align="center" style="background:#f0f0f0;"|'''1x rxn'''
 
| width ="80pt" align="center" style="background:#f0f0f0;"|'''8x rxn mix'''
'''A-tailing'''
|-
Total reaction in 30ul
| 10X End repai buffer||7.00||56.00
|-
A-tailing reaction mix       1x rxn 8x rxn mix
| KAPA End repair enzyme||3.00||24.00
KAPA 10X A-tailing buffer     3.00 24.00
|-
KAPA A-tailing enzyme         2.00 16.00
| H2O||13.00||104.00
H2O                       25.00 200.00
|-
==================================================
| Total||23.00||184.00
Total                       30.00 240.00
|}
Aliquot to column #2: 30ul
* Aliquot to column #1: 23ul
 
== A-tailing ==
'''Adapter ligation'''
A-tailing reaction mix: Total 30ul/reaction<br>
Total reaction in 40ul  
{| {{table}}
Ligation reaction mix 30ul
| width ="150pt" align="center" style="background:#f0f0f0;"|'''A-tailing reaction mix'''
Methylation adapter  10ul
| width ="80pt" align="center" style="background:#f0f0f0;"|'''1x rxn'''
| width ="80pt" align="center" style="background:#f0f0f0;"|'''8x rxn mix'''
  Ligation reaction mix         1x rxn 8x rxn mix
|-
KAPA 5X Ligation buffer         8.00 64.00
| KAPA 10X A-tailing buffer||3.00||24.00
KAPA DNA ligase                 3.00 24.00
|-
H2O                       19.00 152.00
| KAPA A-tailing enzyme||2.00||16.00
Total                       30.00 240.00
|-
| H2O||25.00||200.00
'''Methylation adapter preparation'''
|-
''Alan's note:''  
| Total||30.00||240.00
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
* Aliquot to column #2: 30ul
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)
== Adapter ligation ==
Adapter ligation mix: Total 40ul/reaction -> 10ul is methylation adapters
Methylation adapter 2ulx 8rxn 16.00
{| {{table}}
H2O                 8ulx8rxn 64.00
| width ="150pt" align="center" style="background:#f0f0f0;"|'''Ligation reaction mix'''
==================================================
| width ="80pt" align="center" style="background:#f0f0f0;"|'''1x rxn'''
Total                         80.00
| width ="80pt" align="center" style="background:#f0f0f0;"|'''8x rxn mix'''
Aliquot to column #3: 30ul
|-
20C for 15min
| KAPA 5X Ligation buffer||8.00||64.00
 
|-
'''Reagent preparation'''
| KAPA DNA ligase||3.00||24.00
'''Reagents'''                     '''1rxn''' '''Min volume in 30mL reservior'''
|-
AMPure beads                     120ul ~1.1mL
| H2O||19.00||152.00
20% PEG/2.5M NaCl     90+50 = 140ul ~1.4mL
|-
80% EtOH                   140x6 = 840ul      ~10.0mL
| Total||30.00||240.00
H2O for elution                         23ul ~1.0mL
|}
* Aliquot to column #3: 30ul
== Methylation adapter preparation ==
'''Alan's note:''' <br>
# 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
# 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)
Prepare adapter 2ulx 8rxn --> 16.00ul<br>
H2O 8ulx8rxn --> 64.00ul<br>
Total 80.00ul<br>
== Reagent preparation ==
{| {{table}}
| width ="150pt" align="center" style="background:#f0f0f0;"|'''Reagents'''
| width ="80pt" align="center" style="background:#f0f0f0;"|'''1rxn'''
| width ="120pt" align="center" style="background:#f0f0f0;"|'''Min volume in 30mL reservior'''
|-
| AMPure beads||120ul||~1.1mL
|-
| 20% PEG/2.5M NaCl||90+50 = 140ul||~1.4mL
|-
| 80% EtOH||140x6times||~10mL
|-
| H2O for elution||22ul||~1mL
|}


==epMotion run ==
==epMotion run ==

Revision as of 00:08, 4 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 (except ligation 50ul -> 40ul total)

End repair

Total reaction 70.00 ul
DNA 47.00 ul
End repair reaction mix 23.00 ul


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

A-tailing reaction mix: Total 30ul/reaction

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

Adapter ligation mix: Total 40ul/reaction -> 10ul is methylation adapters

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
  • Aliquot to column #3: 30ul

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)

Prepare adapter 2ulx 8rxn --> 16.00ul
H2O 8ulx8rxn --> 64.00ul
Total 80.00ul

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 140x6times ~10mL
H2O for elution 22ul ~1mL

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