Question

Question S You are studying a gene that is located within a repressed region of chromosome 3, but is expressed following treatment with estradiol. You perform a ChiP-seq experiment with antibodies that detect the fallowing antigens: Estrogen Receptor a, Histone 3 trimethylated at lysine 27 (a repressive chromatin mark), Histone 3 trimethylated at lysine 36 (a de-repressing chromatin mark), RNA polymerase Il. On the lines below, draw the expected ChIP-seq read patterns. (8 points D ERE I Transcriptional start site I Exon ER I PolyA signal sequence H3K27me3 H3K36me3 Polll

Answer 1

ChIP was performed using the Simple ChIP-enzymatic Chromatin IP Kit. Briefly, cells were diluted to 5 × 106 cells/ml in the cell culture medium, and 37% formaldehyde was added to a final concentration of 1% to cross-link histone and DNA. After a 10-min incubation at room temperature, formaldehyde was quenched by adding glycine to a final concentration of 125 mm. Cells were then pelleted and washed with cold PBS twice. 5 × 106 cells were resuspended in 500 μl of micrococcal nuclease buffer and digested with 2000 units of the enzyme for 20 min at 37 °C. Nuclei were pelleted by centrifugation at 13,000 rpm for 1 min and resuspended in 1 ml of ChIP buffer. Nuclear membrane was disrupted by brief sonication (two sets of 10-s pulses), and lysates were clarified by centrifugation at 10,000 rpm for 10 min. The supernatant was used for chromatin immunoprecipitation. The ChIP antibodies were purchased from Abcam.Abca were as follows: H3K4me3 (ab1012), H3K27me3 (ab6002), H3K9me3 (ab8898), H3K36me3 (ab9050), and H3K9ac (ab12179). Ten μg of antibody was used for each immunoprecipitation. Antibodies were incubated with the sample at 4 °C overnight with rotation. After the addition of beads, samples were incubated for another 2 h at 4 °C with rotation. After washing, the immunoprecipitated chromatin was eluted from the bead, and the cross-link was reversed by proteinase K digestion. DNA was then purified using spin columns and quantified. For ChIP-Seq, the library was constructed using Illumina's Chip Sequencing sample preparation kit according to the manufacturer's instructions. Briefly, 10 ng of ChIP-enriched DNA was used for each library construction. First the DNA fragments were repaired to phosphorylated blunt ends using T4 DNA polymerase, Klenow polymerase, and T4 polynucleotide kinase. After DNA fragments were purified using the Qiagen PCR purification kit , an “A” base was added to the 3′-end of the blunt DNA fragment by Klenow fragment (3′ to 5′ exo-) at 37 °C for 30 min. The product was purified by the MinElute purification kit .Sequencing adapters were ligated to the ends of DNA fragments using DNA ligase at room temperature for 15 min, followed by purification with the MinElute PCR purification kit. The product was then separated in 2% agarose gel to remove excess adaptors and to select a size range of library. The fragments with a size range from 150 to 250 bp were excised and purified using the QIAquick gel extraction kit .The library was then amplified by limited PCR (16 cycles) using primers provided by the kit. The concentration and distribution of the library were determined by Agilent Bioanalyzer 2100. The library was sequenced by an Illumina HiSeq2000 system at the Tufts University Genomic Core Facility.