Instrument: Illumina Genome Analyzer
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: SINGLE
Construction protocol: Worm_L3_extraction_vPK1. Worms are frozen, ground, and crosslinked for 10 minutes in 1% formaldehyde. Formaldehyde is quenched and cross-linked tissue washed, then resuspended in FA buffer and subjected to sonication in Bioruptor (14 pulses of 30 seconds with 1 minute rests in between). Extracts are then spun down and soluble fraction is stored for quality tests and future ChIP. Worm_chromatin_immunoprecipitation_vIL2. Appropriate amount of extract is incubated overnight with a proper amount of antibody (exceptional antibodies due to better results are incubated 2hrs). Afterwards, 40ul of equilibrated magnetic beads (either protein A or G, depending on antibody) are added and incubated for 2 hrs. Later, washes with FA, 500mM-salt FA, 1M salt FA, TEL, and TE buffer are performed and DNA is eluted in elution buffer (1% SDS in TE with 250 mM NaCl) ? two times with 57 ?l volume each, at 65°C. Samples are treated with RNAse, proteinase K and then crosslinks are reversed overnight at 65°C. DNA is purified on Qiagen PCR purification columns, tested by q-PCR for ChIP quality, and stored at -20°C for future applications. ChIP or input DNA is blunt ended, A-tailed, ligated to adaptors, amplified by PCR, then size selected using AMPure beads. The protocol uses the Illuimina TruSeq DNA Sample Prep Kit. Prepared sample are sequenced using Illumina GAII or HiSeq2000 at the High Throughput Sequencing Facility of University of North Carolina at Chapel Hill or Cambridge.