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 Pc-TF Genomics
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01/25/13
- Illumina ChIP-seq prep: input DNA
Illumina ChIP-seq prep
> Follow manufacturer's protocol, with some modifications
> Sample (dated 12/04/10):
- "29, FTrx fx Input" (plain cells)
Perform End Repair
Note: forgot to dilute Klenow 1:5 with water before use
| Reagent |
Volume |
Master Mix
|
| Input 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
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--> Aliquot 20 μL master mix, add 30 μL ChIP DNA
--> 20°C/ 30 min. (temp block)
--> 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
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| Klenow exo |
1.0 |
6.0
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50 μL
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--> 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 |
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| DNA ligase buffer |
15.0 |
90.0
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| Adapter oligo mix |
1.0 |
6.0
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| DNA ligase |
4.0 |
24.0
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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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Pc-TF Genomics
