Noi/NOTES/2014-2-3

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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.