Instrument: Illumina HiSeq 2000
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: SINGLE
Construction protocol: Frozen tissues were dissected at -20ºC, thawed quickly and homogenized. Between 20mm3 and 0.5cm3 was used depending on tissue availability. Tissue was immediately chemically cross-linked by the addition of 2ml one-fifth volume of fresh 11% formaldehyde solution in medium and incubated for 10 minutes while rotating at room temperature. Cells were washed with PBS at 4ºC.Cells were lysed and sonicated. The IP was incubated overnight at 4°C with 50 ul of Dynal Protein G magnetic beads (Invitrogen lot# 90513840) that had been pre-incubated with 5 µg of the appropriate antibody for at least 3 hrs in PBS/0.5% BSA at 4°C. Beads were washed 4 times with RIPA buffer and 1 time with TE containing 50 mM NaCl. Bound complexes were eluted from the beads in elution buffer (50 mM Tris pH 8, 10 mM EDTA, 1% SDS) and at the same time crosslinking was reversed by overnight incubation at 65°C. After removal of the beads immunoprecipitated DNA was diluted 1:1 with TE and then treated with RNAse A for 2 hours at 37°C, followed by proteinase K treatment for 2 hours at 50°C. DNA was purified using two consecutive phenol:chloroform extractions using MaXtract High Density gel tubes. The resulting DNA was used for analysis either on the solexa sequencer (HiSeq 2000 genome sequencer) or the SOLiD sequencer (5500xl /550xlW). The sample prep was preformed by using the sequencer corresponding ChIP-Seq sample prep kit according to the manufacturers protocol. HiSeq samples were prepped and run by the MIT BioMicro Center (http://openwetware.org/wiki/BioMicroCenter) using the HiSeq 2000 genome sequencer (10 ChIPs multiplexed per lane). Solexa samples were prepped and run in-house using the SOLiD 5500xl or SOLiD 5500xlW.