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
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