AlanFung:LabNotes/CTCF/2011-6-28

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N9-MDA Bisulfite Converted Sequencing Library Consturction

Objective

  • Work on sperm/ESCs control cell samples plus GM12878 gDNA
  • Sample List
  1. A1-100 sperm cells
  2. A2-100 sperm cells (repeat)
  3. B1-100 ESCs
  4. B2-100 ESCs (repeat)
  5. C1-600pg GM12878 gDNA
  6. C2-600pg GM12878 gDNA>Noi
  7. C3-600pg GM12878 gDNA>Noi
  8. D-1.2ng GM12878 gDNA
  9. E-NTC

Overview

  • Proteinase K Digestion
  • Bisulfite conversion
  • MDA
  • EtOH precipitation
  • Nextera Library Construction (Low input protocol)
  • Page Analysis
  • Sanger Sequencing

Protocol

Proteinase K Digestion

  • Preparation of Proteinase K
  • Add 260 µl (D5020) of Proteinase K Storage Buffer to the tube containing Proteinase K. Dissolve completely
  • Label tubes A1, A2, B1, B2
  1. Sperms: ~50cell/tube; 15 tubes
  2. ES cells: ~50cell/tube; 15 tubes
  • Thaw 4 tubes on ice, we will use 100cells per reaction
  • Spin down tubes and add 9ul nuclease free water and transfer cells to 0.2ml tubes
Content Volume per Reaction
M-Digestion Buffer (2X) 10
Sample 9
Proteinase K 1
Total 20
  • Incubate the samples at 50C for 20min

Bisulfite Conversion

  • Dilute GM12878 gDNA from 50ng/ul to 0.5ng/ul (1:100)
  • Setup two more 600pg GM12878 gDNA reactions for Noi to perform capture using the 330k probe set
0.5ng/ul GM12878 gDNA Volume Per Reaction Make up to 20ul with H2O
600pg 1.2 18.8
1.2ng 2.4 17.6
  • Add 130ul CT Conversion Reagent to all samples
  • Perform reaction in thermocycler
  1. Step1 98C, 8m
  2. Step2 64C, 3.5hr
  3. Step3 4C, storage for up to 20 hr
  • Proceed with Zymo column based method with improvement tweak protocol
  • Elute with 11ul EB Buffer
  • Transfer bisulfite converted samples to 0.2ml tubes for MDA

Heat Denature

  • 92C for 30 sec
  • Transfer to ice immediately

MDA

  • Prepare MDA Master Mix
Content Volume Per Reaction MM
10X RepliPhi Buffer 2 22
1mM N9 Primer 1 11
25mM dNTP 0.8 8.8
RepliPhi Phi29 (100U/ul) 1 11
Klenow Exo Minus (10U/ul) 0.5 5.5
H2O 4.7 51.7
Total 10 110
  • Add 10ul of MDA mastermix to each sample
Plate Layout Plate Layout Plate Layout
1 2
A A1-100 Sperm Cells Noi-600pg GM12878
B A2-100 Sperm Cells Noi-600pg GM12878
C B1-100ESCs Empty
D B2-100ESCs Empty
E C-600pg GM12878 Empty
F D-1.2ng GM12878 Empty
G E1-NTC Empty
H E2-NTC(MDA) Empty
Plate Layout Plate Layout Plate Layout

30C 60 min, 85C 3 min, 4C hold

Ethanol Precipitation

  • Add 2.5X 100% Ethanol
  • Add 0.1X 3M NaOAC
  • Add 1ul glycoblue
  • Store at -80C for 30min (CONTINUE ON 6/29)
  • Spin down at 10,000rpm at 4C for 15min
  • Remove all liquid
  • Add 500ul 4C chilled 75% ethanol
  • Spin at 10,000rpm at 4C for 5 min
  • Remove all liquid
  • Let dry in hood for 5 min
  • Add 8ul of nuclease free H2O (aliquot 2ul to perform PCR Analysis)
  • Let sit at RT to dissolve the pellet

Nextera Library Construction (Low input protocol)

  • In a qPCR tube, mix
  • 3ul genomic DNA
  • 1ul 1:5 Nextera illumina compatible transposomes (10 μl Nextera enzyme, 20 μl 1XTE, 20 μl 100% glycerol)
  • 1ul 5X NExtera HMW buffer
  • Incubate at 55C for 5 min
  • Setup PCR Reaction with Klenow Exo- but without primers
Content Volume Per Reaction MM
Water 17.5 154
DNA 5 Do not add to MM
KAPA Supermix 25 220
Klenow Exo Minus 0.5 4.4
10uM Orange Primer 1 Do not add to MM
10uM Blue Primer 1 Do not add to MM
Total 50 422.4
  • Add 43ul MM to each reaction
  • Incubate reactions at RT for 5 min
  • Put the tubes back on ice
  • Add Primers
  • Perform thermal cycling
Perform Kapa PCR Reaction
95C 3m 
95C 10s
58C 30s
72C 3m
plate read
Goto step 2 repeat 34
72C 10m
Hold 15C

PAGE Analysis

Sanger Analysis