Blog

Experimental design, materials and methods == == 2 . 1 . H2B, H3 and H4 are present in the fly sperm nucleus. We also find evidence intended for the retention of nucleosomes with specific histone H3 trimethylation marks characteristic of chromatin repression (H3K9me3, H3K27me3) and active transcription (H3K36me3). Raw and processed data from the experiments are available at GEO, accessionGSE52165. == 1 . Direct link to deposited data == http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE52165. == 2 . Experimental design, materials and methods == == 2 . 1 . Fly strains used in this study == Two diverse transgenicD. melanogasterw1118 strains were used; one carrying H2Av tagged with red fluorescent protein (H2Av-mRFP1[1],[2]and the other carrying protamine L-Tryptophan tagged with green fluorescent protein (protamine-eGFP;[3]). == 2 . 2 . Isolation of spermatozoa == To avoid any somatic cell contamination, large, fully developed, young males with full seminal vesicles were selected as the source of sperm. Males were anaesthetized with CO2, placed on a clean microscope slide, immersed in a drop of phosphate buffered saline (PBS) and dissected using fine forceps. The accessory glands were separated from the testes, which were then transferred onto a new slide. One end of the seminal vesicle was anchored with forceps and pressure was gently applied with a second pair of forceps to force out Eng the sperm, which were collected as a filamentous bundle in a freezing medium (Dulbecco’s Modified Eagle Medium (DMEM; GIBCO containing 10% dimethylsulfoxide (DMSO) and 20% fetal bovine serum) and stored at 80 C after snap-freezing on dry ice. Arbitrary samples of sperm were stained by DAPI and examined by fluorescence microscopy to assess the quality of the sperm isolation and confirm somatic cell removal. == 2 . three or more. Chromatin immunoprecipitation (ChIP) == For each ChIP replicate, 106frozen spermatozoa were thawed at room heat, washed in cold PBS and fixed in 100 l 1% formaldehyde in PBS at room temperature L-Tryptophan intended for 10 min. Crosslinking was quenched by the addition of 400 l 0. 125 M glycine in PBS and incubation L-Tryptophan at 4 C intended for 5 min. Cells were washed twice in 500 l ice-cold PBS and incubated intended for 1 h at room temperature in 500 l lysis buffer A (10 mM DTT, 10 mM HEPES pH 8, 10 mM EDTA, 0. 5 mM EGTA, 0. 25% Triton X-100, 10 mM DTT, 0. 5% (w/v) SDS and protease inhibitors [10 mg/ml aprotinin, 5 mg/ml leupeptin, and 0. 5 mM PMSF]). The DTT was quenched by the addition of N-ethylmaleimide to a final concentration of 5 mM and the cells were resuspended in 100 l lysis buffer B (10 mM HEPES pH 8, 200 mM NaCl, 0. 5 mM EGTA, 1 mM EDTA, 0. L-Tryptophan 01% Triton X-100 and protease inhibitors), pelleted and resuspended in 100 l IP buffer (25 mM Tris pH 8, 150 mM NaCl, 2 mM EDTA, 1%Triton X-100 and 7. 5% glycerol) with 0. 25% SDS and protease inhibitors. Chromatin was sheared to approximately 500 bp fragments by sonication on ice using a MEM Soniprep 150 sonicator at 6 micron amplitude for 5 cycles of 20 second sonication and 40 second cooling. Lysates were diluted in four volumes I of P buffer and cleared by centrifugation. For each IP, a 10 l suspension of Protein G Dynabeads (pre-absorbed with BSA and herring sperm DNA) was incubated in 500 l 100 mM sodium phosphate pH 8, 0. 5% BSA, that contains a 5 g ChIP-grade antibody (or 5 g goat anti-rabbit IgG antibody for mock IPs) at 4 C with rotation for 2 h. The Dynabeads were washed in IP buffer and resuspended in 490 l cleared lysate along with 25 g sonicated herring sperm DNA. Reactions were incubated at 4 C with rotation intended for 4 h. Immune complexes were recovered, suspended in 500 l buffer 1 (20 mM TrisHCl pH 8, and 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0. 1% SDS and protease inhibitors) and incubated on ice intended for 10 min before being washed twice in 500 l buffer 2 (20 mM TrisHCl pH 8, 500 mM NaCl,.