Noi/NOTES/2014-2-3
Jump to navigation
Jump to search
WGBS library preparation of low input DNA (200ng gDNA) of 16 sample from Dr. Brinda Rana's lab[edit]
- I have picked up 16 gDNA samples from Dr. Rana on 2013-10-21 and re-quantified concentration by Qubit dsDNA HS assay.
Orignal sample info from Dr. Rana[edit]
Tube # | ID# | PairID | Time | Case/Control | quantity (microgram, ug) | volume (ul) |
1 | 27499 | Twin 1 A | V1 | control | 1ug | 2 |
2 | 27816 | Twin 1 B | V1 | case | 1ug | 2.5 |
3 | 28593 | Twin 3 A | V1 | control | 1ug | 2.5 |
4 | 29006 | Twin 3 B | V1 | case | 1ug | 2.5 |
5 | 36452 | Twin 5 A | V1 | case | 1ug | 1 |
6 | 36453 | Twin 5 B | V1 | control | 1ug | 1.5 |
7 | 30603 | Twin 6 A | V1 | control | 1ug | 1.5 |
8 | 30604 | Twin 6 B | V1 | case | 1ug | 2 |
9 | 38951 | Twin 7 A | V1 | control | 1ug | 1 |
10 | 38952 | Twin 7 B | V1 | case | 1ug | 2 |
11 | 39427 | Twin 8 A | V1 | case | 1ug | 1.5 |
12 | 39428 | Twin 8 B | V1 | control | 1ug | 2 |
13 | 29586 | Twin 2 A | V1 | case | 1ug | 2 |
14 | 32466 | Twin 2 B | V1 | control | 1ug | 1 |
15 | 33643 | Twin 4 A | V1 | case | 1ug | 3 |
16 | 33644 | Twin 4 B | V1 | control | 1ug | 2.5 |
MZ Twin Discordant for Smoking Brinda Rana bkrana@ucsd.edu
- Initial volume of DNA was very low ~2-3ul in 1.5mL tube. I then transferred sample to strip tube and added 20ul TE buffer and used 1ul for Qubit quantification.
Qubit dsDNA HS quantification[edit]
Sample | Conc. in the Qubit | ' | uL used | Dilution | Sample Conc. (ng/ul) | Yield in 20ul (ng) |
BR_1 | 109 | ng/mL | 1 | 200 | 21.70 | 434.00 |
BR_2 | 270 | ng/mL | 1 | 200 | 54.00 | 1080.00 |
BR_3 | 287 | ng/mL | 1 | 200 | 57.30 | 1146.00 |
BR_4 | 360 | ng/mL | 1 | 200 | 71.90 | 1438.00 |
BR_5 | 198 | ng/mL | 1 | 200 | 39.50 | 790.00 |
BR_6 | 147 | ng/mL | 1 | 200 | 29.40 | 588.00 |
BR_7 | 169 | ng/mL | 1 | 200 | 33.70 | 674.00 |
BR_8 | 191 | ng/mL | 1 | 200 | 38.30 | 766.00 |
BR_9 | 161 | ng/mL | 1 | 200 | 32.10 | 642.00 |
BR_10 | 120 | ng/mL | 1 | 200 | 24.00 | 480.00 |
BR_11 | 201 | ng/mL | 1 | 200 | 40.30 | 806.00 |
BR_12 | 231 | ng/mL | 1 | 200 | 46.30 | 926.00 |
BR_13 | 139 | ng/mL | 1 | 200 | 27.80 | 556.00 |
BR_14 | 147 | ng/mL | 1 | 200 | 29.50 | 590.00 |
BR_15 | 176 | ng/mL | 1 | 200 | 35.20 | 704.00 |
BR_16 | 195 | ng/mL | 1 | 200 | 39.00 | 780.00 |
Covaris gDNA shearing (2014-01-30)[edit]
- Sample preparation for gDNA shearing
Sample | Sample conc. (ng/ul) | Yield in 20ul (ng) | Volume for 200ng (ul) | H2O (ul) |
BR_1 | 21.70 | 434.00 | 9.22 | 40.78 |
BR_2 | 54.00 | 1080.00 | 3.70 | 46.30 |
BR_3 | 57.30 | 1146.00 | 3.49 | 46.51 |
BR_4 | 71.90 | 1438.00 | 2.78 | 47.22 |
BR_5 | 39.50 | 790.00 | 5.06 | 44.94 |
BR_6 | 29.40 | 588.00 | 6.80 | 43.20 |
BR_7 | 33.70 | 674.00 | 5.93 | 44.07 |
BR_8 | 38.30 | 766.00 | 5.22 | 44.78 |
BR_9 | 32.10 | 642.00 | 6.23 | 43.77 |
BR_10 | 24.00 | 480.00 | 8.33 | 41.67 |
BR_11 | 40.30 | 806.00 | 4.96 | 45.04 |
BR_12 | 46.30 | 926.00 | 4.32 | 45.68 |
BR_13 | 27.80 | 556.00 | 7.19 | 42.81 |
BR_14 | 29.50 | 590.00 | 6.78 | 43.22 |
BR_15 | 35.20 | 704.00 | 5.68 | 44.32 |
BR_16 | 39.00 | 780.00 | 5.13 | 44.87 |
- Program's name at GMI (contacted Rebecca Orlando): zhang_400bp_tube.xx
Followed up the set up for Covaris shearing from previous experiment [[1]] Target BP 400 Tube Micro tube Duty Factor 10% Peak Incident Power (w) 140 Cycle per Burst 200 Time (secs) 55 Volume 50ul Temperature (c) 7 Intensifier Yes
- Verify sheared gDNA in 6% TBE gel
File:ZhangLab 2 2014-02-03 11hr 37min shearedgDNA 1-8.jpg File:ZhangLab 2 2014-02-03 11hr 44min shearedgDNA 9-16.jpg
- Note that I planned to load1.5ul of sheared DNA as was performed for N37 samples. I accidentally mix loading dye with 80% EtOH, so samples were floating above the well. I then re-prepared samples and used 1ul to load in the gel. Therefore I have sheared DNA left ~47ul for library prep.
Preparation of enzyme mix for End repair, A-tailing, Ligation, and Methylation adapters[edit]
- Note
- I followed the same protocol for low input WGBS, but made slightly change that will we noted here.
- I could not use epMotion to process these samples because Window could't start up. I have tried to open many time, but finally it showed black screen. It said that the possibility could be hardware or software changed. I saved error message and will ask Alan help when he is back.
End repair[edit]
Sheared gDNA 200ng in 47ul
Total reaction | 70 | ul |
DNA | 47 | ul |
End repair reaction mix | 23 | ul |
KAPA End repair mix | 1x rxn | 17x rxn mix |
10X End repai buffer | 7.00 | 119.00 |
KAPA End repair enzyme | 3.00 | 51.00 |
H2O | 13.00 | 221.00 |
Total | 23.00 | 391.00 |
- Aliquot to column #1: 48ul
- Add 23ul of End-repair reaction mix - Mix by pipetting 20 times - Shake at 1000 rpm for 5min (lid on). SKIPPED - Incubate at 20C for 30min - Add 120ul of diluted AMPure beads - Mix at 750rpm for 5min, 25C (lid on) SKIPPED - Incubate at 25C for 8 min - Put on magnet and discard spnt - Wash twice with 140ul 80% EtOH - Dry the beads
A-tailing[edit]
- Total 30ul/reaction
A-tailing reaction mix | 1x rxn | 17x rxn mix |
KAPA 10X A-tailing buffer | 3.00 | 51.00 |
KAPA A-tailing enzyme | 2.00 | 34.00 |
H2O | 25.00 | 425.00 |
Total | 30.00 | 510.00 |
- Aliquot to column #2: 63ul
- Add 30ul of A-tailing reaction mix - Mix by pipetting 20 times - Shake at 1000 rpm for 5min (lid on) SKIPPED - Incubate at 30C for 30min - Add 54ul of 20%PEG/2.5M NaCl (for standard protocol, added 90ul to 50ul reaction) - Mix at 750rpm for 5min, 25C (lid on) SKIPPED - Incubate at 25C for 8 min - Put on magnet and discard spnt - Wash twice with 140ul 80% EtOH - Dry the beads
Adapter ligation[edit]
Total reaction | 50 | ul |
Ligation reaction mix | 40 | ul |
Diluted Methylation adapters | 10 | ul |
Ligation reaction mix | 1x rxn | 17x rxn mix |
KAPA 5X Ligation buffer | 10.00 | 170.00 |
KAPA DNA ligase | 4.00 | 68.00 |
H2O | 26.00 | 442.00 |
Total | 40.00 | 680.00 |
- Aliquot ligation reaction mix to column #3: 84ul
Methylation adapter preparation[edit]
- Order Methylation Adapter Oligo kit from Illumina (cat #ME-100-0010)
- 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 0.2ug of DNA, used 0.4ul of adapters, but dilute to final volume 10ul (This volume is larger than KAPA protocol, but the total amount is the same)
- Previously, I did wrong calculation. I used 0.2ul, but it worked well.
Prepare adapter | 0.4ulx 18rxn | 7.20ul |
H2O | 9.6ulx18rxn | 172.80ul |
Total | 10.00ul | 180.00ul |
- Aliquot diluted methylated adapter to column #4: 22ul
- Add 40ul of Ligation reaction mix - Mix by pipetting 10 times - Add 10ul of diluted Methylation adapters - Mix by pipetting 10 times - Shake at 1000 rpm for 5min (lid on) SKIPPED - Incubate at 20C for 15min - Add 50ul of 20%PEG/2.5M NaCl - Mix at 750rpm for 5min, 25C (lid on) SKIPPED - Incubate at 25C for 8 min - Put on magnet and discard spnt - Wash twice with 140ul 80% EtOH - Dry the beads - Resuspend adapter ligated DNA with ~23ul EB buffer
qPCR to check if the end repair, A-tailing, and adapter ligation work[edit]
- I only tried some samples (#1-4, 9-11)
Components | 1rxn | 8.5 rxn mix |
Adapter ligated DNA | 1.00 | 0.00 |
10uM PCR_F | 0.50 | 4.25 |
10uM PCR_R.Ind2 | 0.50 | 4.25 |
2x KAPA SYBG MM | 12.50 | 106.25 |
H2O | 10.50 | 89.25 |
Total | 25.00 | 212.50 |
- Aliquot 24ul, add 1ul of adapter ligated DNA
Program 98C 2min --> (98C 15sec -> 64C 20sec -> 72C 30sec) x 12 cycles --> 72C 3min --> 15C Hold
- qPCR didn't work. This was very unlikely since most of the time even I started with lower input (as low as 2.5ng), it always worked fine for regular library prep. I was pretty sure that I prepared master mix right. There could be two reasons for this failure. One is I used expired AmPure beads that might cause poor recovery. The other one is methylated adapters were different from the previous one that I used. I tried to check sequences of this adapter, but it was not provided in any source.
- I repeated qPCR using 2ul of template and iQ MM as I used before N37 low input experiment and amplified for 15 cycles. I loaded 5ul of PCR product in the gel and could see amplification. This mean that adapter and ligation worked. However, the efficiency was very low compare to previous experiment. I suspect the bead that I used since it was expired. I will repeat experiment since I still have enough gDNA for one or two rounds.