GSM1201947: input DNA; Homo sapiens; ChIP-Seq
20 million CD4+ T cells were used in total for the ChIP-seq experiments. Cells were cross-linked by the addition of 1% of formaldehyde for 10 minutes at room temperature.  Before ChIP, cells were lysed and sonicated. For sonication, lysates were treated with Triton X-100 to 1% and then subjected to 20 cycles of 20 seconds sonication followed by 60 second rest in a Bioruptor UCD-200 (Diagenode). The supernatant was incubated for 24 hrs at 4°C with 20 μl of Dynabead Protein G beads (Invitrogen) which had been conjugated with 10 μg of the REST antibody (Millipore 07-579). After ChIP, samples were washed with low salt, high salt, LiCl and Tris-EDTA buffer for 5 min. Bound complexes were eluted and crosslinking was reversed by overnight incubation at 65°C. Whole cell extract DNA was also treated for crosslink reversal. Purified immunoprecipitated DNA was prepared for sequencing following the guidelines of Illumina library preparation kit. In short, DNA was end-repaired using a (End-It DNA Repair Kit, Epicentre). A-tails were added to the blunt ends and Adaptor Oligo Mix (Illumina) was used in the ligation step using LigaFast Kit (Promega). Following adapter ligation, DNA was PCR amplified for 25 cycles and agarose gel extraction was used to isolate library fragments of 150-300 bp. The purified DNA was captured on an Illumina flow cell. Libraries were sequenced on an Illumina HiSeq 2000 following the manufacturer's protocols.