GSM1641318: input DNA; Homo sapiens; ChIP-Seq
Cells were fixed using formaldehyde solution, lysed and sonicated to obtain chromatin fragments of 100-500bp size. The sonicated chromatin was then subjected to immunoprecipitation with polyclonal anti-STAT3 antibody (rabbit polyclonal αSTAT3 3291L, Cell Signaling Technology). The STAT3 bound chromatin was isolated using Dynal magnetic beads coated with secondary antibody anti-rabbit IgG (Dynal Biotech). The immunoprecipitated DNA was eluted, reverse crosslinked and purified using Qiagen minielute columns. The sample preparation for Solexa sequencing was performed according to Illumina's recommendations by Illumina service provider, Fasteris Life Sciences, Switzerland. Briefly, the ChIP DNA was end-repaired with T4 DNA polymerase, Klenow and PNK, and after purification with Qiagen minielute columns, a 3-´A was added using Klenow (exo-). The -A tailed DNA samples were purified and ligated with Illumina paired ends adaptors. After adapter ligation, fragments of size range of 200-300bp were purified from agarose gel and subjected to PCR amplification using Phusion polymerase with paired ends primers for 18 cycles. The libraries were quantified using PicoGreen Assay (Q-bit Invitrogen) before in situ amplification on the Illumina Cluster Station to generate DNA colonies according to the manufacturer's standard recommended protocol. Sequencing was performed on an Illumina Genome Analyzer “GAII” for 36 cycles using sequencing kit V-1.0 with scanning buffers V-2.0 and GA data analysis pipeline 1.0rc4. Each library was sequenced in a single read channel that produced between 10 to15 million reads. The sample preparation for Solexa sequencing was performed according to Illumina's recommendations by Illumina service provider, Fasteris Life Sciences, Switzerland. Briefly, the ChIP DNA was end-repaired with T4 DNA polymerase, Klenow and PNK, and after purification with Qiagen minielute columns, a 3-´A was added using Klenow (exo-). The -A tailed DNA samples were purified and ligated with Illumina paired ends adaptors. After adapter ligation, fragments of size range of 200-300bp were purified from agarose gel and subjected to PCR amplification using Phusion polymerase with paired ends primers for 18 cycles. The libraries were quantified using PicoGreen Assay (Q-bit Invitrogen) before in situ amplification on the Illumina Cluster Station to generate DNA colonies according to the manufacturer's standard recommended protocol. Sequencing was performed on an Illumina Genome Analyzer “GAII” for 36 cycles using sequencing kit V-1.0 with scanning buffers V-2.0 and GA data analysis pipeline 1.0rc4. Each library was sequenced in a single read channel that produced between 10 to15 million reads.