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
10 mM 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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