Noi/NOTES/2012-8-1: Difference between revisions
Jump to navigation
Jump to search
>Noi |
>Noi |
||
Line 87: | Line 87: | ||
* Performed bisulfite conversion manually and followed the product's protocol: [[Media:EZ-96DNAMethylation-LightninMagPrep.pdf| EZ-96DNAMethylation-LightninMagPrep.pdf]] | * Performed bisulfite conversion manually and followed the product's protocol: [[Media:EZ-96DNAMethylation-LightninMagPrep.pdf| EZ-96DNAMethylation-LightninMagPrep.pdf]] | ||
* Elute with elution buffer 40ul | * Elute with elution buffer 40ul | ||
== ssDNA Qubit quantification == | |||
* Used 3ul of bis-cvt DNA for the assay (dilution 200/3) | |||
== Amplification of bisulfite sequencing llibraries (Test) == | == Amplification of bisulfite sequencing llibraries (Test) == |
Revision as of 01:14, 4 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 (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:
- 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
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:
Bisulfite conversion using EZ-96 DNA Methylation-Lightning™ MagPrep
- Performed bisulfite conversion manually and followed the product's protocol: EZ-96DNAMethylation-LightninMagPrep.pdf
- Elute with elution buffer 40ul
ssDNA Qubit quantification
- Used 3ul of bis-cvt DNA for the assay (dilution 200/3)
Amplification of bisulfite sequencing llibraries (Test)
- Test if the adapter ligation reaction worked before amplify the libraries with barcoded primers
Components | 1rxn | 10 rxn mix |
DNA template (1:9 diluted) | 2.50 | 0.00 |
10uM PCR_F | 0.50 | 5.00 |
10uM PCR_R.Ind1 | 0.50 | 5.00 |
2x iQ MM | 12.50 | 125.00 |
H2O | 9.00 | 90.00 |
Total | 25.00 | 250.00 |
- Aliquot 22.5ul, add 2.5ul of bis-cvt DNA template (2.5 out of 40ul)
Program 98C 2min (98C 15sec -> 64C 20sec -> 72C 30sec) x15cycles -> 72C 3min -> 15CHold
qPCR results
- Will be added later
- Loaded 2.5 ul of bis-cvt DNA (#1 & #4) and 2.5ul of amplified amplicons
File:ZhangLab 2 2012-08-02 11hr 31min-HAPMAP-bis-test.jpg
Amplification of bisulfite sequencing libraries with N2 barcoded primers
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 |
qPCR results
- Will be added later
- 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