AlanFung:LabNotes/Methylome/2012-1-17

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GM12878 Library[edit]

Sample Preparation[edit]

  • Get GM12878 samples from Noi
Took 20ul (250ng/ul) from E2 position of the sample plate, add 30ul ddh2o making final volume to 50ul for shearing
  • Take 5ug of samples and prepare for shearing (Make total volume to 100ul with ddh2o)
  • Covaris shearing of 5ug gDNA to ~400bp
  • Contacted Yang at Kelly Frazer's lab to help me with it (so Yang says they don't have the coavris shearing machine anymore and they shear their samples at Biogem now, emailed James and see if i can get it sheared over there)
  • James helped me out to shear the samples, he used the following settings
Target BP 400
Tube Micro
Duty Factor 10%
Peak Incident Power (w) 140
Cycle per Burst 200
Time (secs) 55
Volume 50ul
Temperature (c) 7
Intensifier Yes
S water level 12
E water level 6

End repair & A-tailing[edit]

End Repair[edit]

Content Volume
Fragmented DNA 50
End Reapir buffer 10
End Repair Enzyme Mix 5
ddH2O 35
Volume 100
  • incubate at 20C for 30mins
  • Purify DNA with Qiaquick column

A-tailing[edit]

Content Volume
End repaired blunt dna 30
A-tailing reaction buffer 10x 5
klenow fragment 3
H2o 12
Total Volume 50
  • Incubate @ 37C for 30mins
  • Qiaquick
  • Keep 1ul for gel image

ligation to methylated adaptor[edit]

  • 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
Content Volume
KAPA 5x ligation buffer 11.43
KAPA DNA Ligase 5.71
Methylation Adapter Oligo 10
A-Tailed DNA 30
Total 57.14
  • Incubate at 20C for 15mins
  • Minelute w 20ul EB Buffer
  • Keep 1ul for gel image

Bisulfite Conversion[edit]

  • Add 20ul of sample to 130ul of CT conversion reagent solution
  • Mix and centrifuge
  • Thermal cycle
    • 98C 8mins
    • 64C 3.5hrs
    • 4C forever
  • Add 600ul M-binding buffer to zymo column and load sample
  • centrifuge at 14000 rpm for 30sec
  • Add 200ul M-Delsulphonation buffer to column, let stand at RT for 15 mins
  • Centrifuge for 30sec
  • Add 200ul M wash buffer to the column
  • Centrifuge for 30sec
  • repeat washing
  • Elute w 10ul warm EB

PCR[edit]

Content Volume
PfuTurboCx Hotstart DNA Polymerase 2.5U/ul 1
10x PfuTurbo reaction buffer 5
dNTPs 1mM each 10
PCR F 10uM 1
PCR R 10uM 1
Template 2
50X SYBR Green 0.2
H2o 29.8
Total 50

95 °C for 2 min, 98 °C for 30 s, then 29 cycles of 98 °C for 15 s, 60 °C for 30 s and 72 °C for 4 min, ending with one 72 °C for 10 min step File:Methylone 120117.jpg

  • run gel to see the amplicon size

File:ZhangLab 2 2012-01-30 16hr 54min.jpg

  • 1-after A-tailing
  • 2-after adapter ligation
  • 3-after bisulfite conversion and PCR
  • Dr. Zhang decides to switch the polymerase to iQ, it could be the SYBR Green that inhibits the amplification

PCR w iQ Supermix[edit]

Content Volume
2X iQ Supermix 10
PCR F 10uM 0.4
PCR R 10uM 0.4
Template 1
H2O 8.2
Total 20
  • 95C 2min
  • 95C 15sec
  • 60C 30sec
  • 72C 30sec
  • Plate Read
  • Go to step2 repeat 19 times
  • 72C 10min
  • 15C hold
  • no amplification after 20 cycles.
  • Possibility of failed amplification
    • Template lost during bisulfite conversion
    • Wrong Primer
    • Incorrect PCR condition

Troubleshooting[edit]

  • According to illumina tech support the adapters come double-stranded
PE Adapters 
5' P-GATCGGAAGAGCGGTTCAGCAGGAATGCCGAG 
5' ACACTCTTTCCCTACACGACGCTCTTCCGATCT
Name in original paper Nick-name Sequence (5\'->3\') Sequence Modification Reference
PCR_F Sol. Amp. (Forward) AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATC*T 3'-Phosphorothioate bond [2]
PCR_R Sol. Amp. (Reverse) CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATC*T 3'-Phosphorothioate bond [2]
  • so either ligation failed or we lost the samples due to bisulfite conversion

Troubleshooting #2 02/02/2012[edit]

TBU Gel[edit]

2. Dilute sample 10x (1uL sample + 4 uL of water) so total is 10uL of sample, and add 5 uL of 2x TBE buffer
3. Denature the DNA with loading dye on thermalcycler 75C for 7 min, quickly transfer to cooling rack, wait for 1 min.
4. Use 6% 10 well TBU gel, add 0.5x TBE buffer, flush the wells very well to remove all urea.
5. Pre-run the gel at 200V for 10 min, then load the samples into the wells, run at 200V for 30 min.  Load the ladder as 8, 4, 2, 1 uL, and load the samples as 10, 5, 2.5 uL.
6. Stain the gel with 2.5 uL SYBR gold in ~100 mL of TBE in clean tray. Place the tray on an orbital shaker for 5 min. Then quantify the gel to calculate the % yield.

File:ZhangLab 2 2012-02-02 15hr 37min.jpg

PCR w Positive Control[edit]

  • Used Alice library w N2 Indx 9 as positive control
Content Volume MM
2X iQ Supermix 10 21
5uM PCR_R_index+PCR_F 0.8 1.68
Template 1 Do not add to template
H2O 8.2 17.22
Total 20 39.9
  • 95C 2min
  • 95C 15sec
  • 60C 30sec
  • 72C 30sec
  • Plate Read
  • Go to step2 repeat 19 times
  • 72C 10min
  • 15C hold

File:ZhangLab 2 2012-02-02 15hr 53min.jpg File:ZhangLab 2 2012-02-02 17hr 38min.jpg