Illumina Sequencing Library Construction from ChIP DNA

Materials and Reagents

1. QIAquick PCR purification kit (QIAGEN, catalog number: 28104 )
   
2. QIAquick gel extraction kit (QIAGEN, catalog number: 28704 )
   
3. MinElute PCR purification kit (QIAGEN, catalog number: 28004 ), store the columns at 4 °C.
   
4. Gibco UltraPure water (Life Technologies, Gibco^®, catalog number: 10977-015 )
   
5. End-it DNA End repair kit (Epicentre^®, catalog number: ER0720 )
   
6. Klenow fragment (3’ ≥ 5’ exo minus) (New England Biolabs, catalog number: M0212S )
   
7. 100 mM dATP (Life Technologies, Invitrogen™, catalog number: 10216-018 or VWR International, catalog number: PAU1201 )
   
8. LigaFast ligation kit (Promega corporation, catalog number: M8221 )
   
9. Ethanol
   
10. Elution buffer
    
11. End repair buffer
    
12. Klenow buffer
    
13. Cyan/orange loading buffer
    
14. 2% E-gel, precast agarose gel (Life Technologies, Invitrogen™) (can be replaced by house made 2% agarose gel)
    
15. Phusion HF PCR master mix (New England Biolabs, catalog number: F531S or F531L )
    
16. Illumina Adapter oligo mix from Illumina sequencing kit, but can be replaced by house made oligo mix using fast denaturation and slow reannealing as described in reference 1. The adapter oligo sequences are:
    5' P-GATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG
    5' ACACTCTTTCCCTACACGACGCTCTTCCGATCT
    
17. Illumina PCR primers 1.1 and 2.1 from Illumina sequencing kit, but can be replaced by ordinary synthesized oligos with the following sequences:
    Illum1.1:
    5' AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCC
    GATCT
    Illum2.1:
    5' CAAGCAGAAGACGGCATACGAGCTCTTCCGATCT
    

Equipment

1. BECKMAN centrifuges and rotor (Beckman Coulter)
   
2. PCR thermocycler (PerkinElmer or F. Hoffmann-La Roche)
   
3. NanoDrop Micro-Volume UV-Vis Spectrophotometer
   

Procedure