Fragment library construction kit

ChIP-seq libraries for all the samples were prepared from around 5 ng of DNA using fragment library construction kit from Life technologies for SOLiD sequencing. Briefly ChIPed DNA samples were end repaired and adapters were ligated using T4 DNA ligase. Ligated DNAs were subsequently amplified using adapter specific bar-coded primers for 15 cycles. DNA purification at every step was performed using Agencourt XP beads (Beckman Coulter). Library profiles were assessed using Agilent bioanalyzer 2100 high-sensitivity DNA kit. Equimolar amount of libraries were pooled and 50 bp reads were sequenced in-house using SOLiD 4.0 sequencer (Applied Biosystems).

The amplicons were first purified with Qiagen PCR MinElute. We then applied the Amplicon Concatenation Protocol 03/2012 from Life Technologies included with the SOLiD Fragment Library Construction Kit. We end-repaired 300 ng of each amplicon, and then purified them with the SOLiD Library Column Purification Kit. The amplicons were then ligated with T4 ligase and purified with the SOLiD Library Column Purification Kit. The concatenated amplicons (100 ng) were then sonicated with the Covaris system (six cycles, 10% duty cycles, intensity 5, 100 cycles per burst, time 60 s). The fragmented DNA was then processed with the Ion Xpress Plus gDNA and Amplicon Library (01/31/2012), with slight modifications. The SizeSelect Gel (from Life Technologies) was cut at 330 bp and the amplification step was performed with eight cycles. Independent samples were pooled in different amounts to achieve different sensitivities and then processed with PCR on the OneTouch system from Life Technologies. The libraries were sequenced on a Ion Personal Genome Machine (PGM) system with 200-bp kit chemistry.

ChIP-seq was performed using the SOLiD 5500 platform (Applied Biosystems). ChIP-seq libraries were prepared from 5 ng of DNA extracted after chromatin immunoprecipitation for different histone modifications using fragment library construction kit from Life technologies. Here, end repairing of the ChIPed DNA was carried out and then adaptors were ligated using T4 DNA ligase. Adaptor ligated products were amplified using adaptor specific primers for 12 cycles. As per the manufacturer’s protocol DNA purification was performed using Agencourt XP beads (Beckman Coulter). Quality and quantity of the libraries were analyzed using Agilent bioanalyzer 2100 high-sensitivity DNA kit. The pooled libraries were used for sequencing.