User:Karmella Haynes/Notebook/PcTF Genomics/2011/12/13

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12/13/11

  • ✓ Illumina ChIp-seq prep: Pc-TF ChIP samples 1-6 (Day one)



Illumina ChIP-seq prep
> Follow manufacturer's protocol, with some modifications
> Samples (dated 12/20/10):

  1. "32, 126-1 myc" (hPc-TF)
  2. "33, 126-1 IgG" (hPc-TF mock)
  3. "36, 132-8 myc" (TF)
  4. "37, 132-8 myc" (TF mock)
  5. "38, FTRx K27m" (plain cells)
  6. "39, FTRx K27m" (plain cells mock)


Perform End Repair
Note: forgot to dilute Klenow 1:5 with water before use

Reagent Volume Master Mix
ChIP enriched DNA 30.0 μL ---
dH2O 10.0 60.0
T4 DNA ligase buffer 5.0 30.0
dNTP mix 2.0 12.0
T4 DNA Polymerase 1.0 6.0
Klenow DNA polymerase 1.0 6.0
T4 PNK 1.0 6.0
  50 μL

--> Aliquot 20 μL master mix, add 30 μL ChIP DNA
--> 20°C/ 30 min. (Xiao's thermal cycler)
--> QIAquick PCR Purification, elute with 34 μL EB


Add 'A' Bases to the 3' End of the DNA Fragments

Reagent Volume Master Mix
DNA sample 34.0 μL ---
Klenow buffer 5.0 30.0
dATP 10.0 60.0
Klenow exo 1.0 6.0
  50 μL

--> Aliquot 16 μL master mix into DNA
--> 37°C/ 30 min. (heat block)
--> Zymo clean and concentrator, elute with 10 μL st.dH2O


Ligate Adapters to DNA Fragments
Note: Did not dilute the adapter oligo mix

Reagent Volume Master Mix
DNA sample 10.0 μL ---
DNA ligase buffer 15.0 90.0
Adapter oligo mix 1.0 6.0
DNA ligase 4.0 24.0
  30 μL

--> Aliquot 20 μL to DNA samples
--> 15 min./ r.t.
--> Zymo clean and concentrator, elute with 10 μL st.dH2O
--> Store at 4°C o/n


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