AlanFung:LabNotes/Methylome/2012-1-17

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Revision as of 18:54, 27 January 2012 by >Alan6017518 (→‎Bisulfite Conversion)
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GM12878 Library

Sample Preparation

  • 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

End Repair

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

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 Y adaptor

  • 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
5X ligation buffer 11.43
DNA Ligase 5.71
Methylation Adapter Oligo 10
A tailed DNA 30
h2o 0
Total 57.14
  • Incubate at room temperature for 30 minutes
  • Minelute w 20ul EB Buffer

Bisulfite Conversion

  • 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 warm EB

PCR

Size selection