The investigated promoter region resulted methylated in MSI H colon tumors only

Total RNA was reverse transcribed using oligo primers and M MLV reverse transcriptase, and was then subjected to real time quantitative PCR using gene specific primers All target gene expression was normalized to TBP ex pression. Each experiment was conducted with a mini mum of three biological replicates. Chromatin immunoprecipitation sequencing ChIP ChIP was performed as described previously JAK3 阻害剤 with a few modifications. AC16 cells were seeded at 3 106 cells per 15 cm diameter plate and treated as described above. The cells were cross linked with 1% paraformalde hyde in PBS for 10 minutes at 37 C and quenched in 125 mM glycine in PBS for 5 minutes at 4 C. The cells were then collected and lysed in Farnham lysis buffer. A crude nuclear pellet was collected by centrifugation, resuspended in lysis buffer, and incubated on ice for 10 minutes.<br><br> The chromatin was sheared at 4 C by sonication supplier LDE225 using a Bior uptor UC200 at the high setting for four 5 minute cycles of 30 seconds on and 60 seconds off to generate chroma tin fragments of 300 bp in length. The soluble chromatin was diluted 1 10 with dilution buffer and pre cleared with protein A agarose beads. The pre cleared supernatant was used in immunoprecipitation reactions with antibodies against the factor of interest or with rabbit IgG as a control. The immunoprecipitated material was washed once with low salt wash buffer, once with high salt wash buffer, once with LiCl wash buffer, and once with 1x Tris EDTA. The immunoprecipi tated material was eluted in elution buffer and was then digested with proteinase K and RNase H to remove protein and RNA, respectively.<br><br> The immunoprecipitated genomic DNA was then extracted with phenol chloroform isoamyl alcohol and pre cipitated with ethanol. ChIP seq library preparation The immunoprecipitated DNA was purified further using the MinElute PCR Purification Kit from Qiagen. After purification, LY2157299 TGF-beta 阻害剤 50 ng of ChIPed DNA for each condi tion was used to generate libraries for sequencing, as previously described, with some modifications. Briefly, the DNA was end repaired and a single A base overhang was added using the Klenow fragment of E. coli DNA polymerase. The A modified DNA was ligated with Illumina sequencing adaptors using the Illumina TruSeq DNA Sample Prep Kit. The ligated DNA was size selected by agarose gel electrophoresis and extraction, amplified by PCR, and purified using AmPure beads.<br><br> The final libraries were subjected to QC and sequenced using an Illumina Hiseq 2000 per the manufacturers instructions. ChIP seq data analyses NF κB p65 and Pol II ChIP Seq data in control and TNF treated AC16 cells were generated in the experi ments described herein. The ChIP seq reads were aligned to the hg19 human reference genome using the Bowtie software package. Mapped reads were further converted to bed files for later Metagene and read density analyses and wiggle files counting reads in non overlapping 200 bp windows across the genome for presentation as gen ome browser tracks by using the BEDTools software package. Global run on sequencing Isolation of nuclei AC16 cells were seeded at 3 106 cells per 15 cm diameter plate and treated as described above.