Illumina HiSeq 3000 paired end sequencing; ChIP-seq of Drosophila melanogaster plasmatocytes agains various histone modifications
ChIP-seq of Drosophila melanogaster plasmatocytes agains various histone modifications
Flies were raised and maintained on standard cornmeal food at 25°C or room temperature. For experiments on wild type OrR, adult fly collections were left to lay eggs on apple juice agar plates with yeast paste overnight at 25°C. After 24h hatched larvae were washed of the plate and further larvae were allowed to hatch for 2h. Then, larvae were picked of the plate and transferred into 36mm food tubes with yeast paste at a density of 120 larvae per tube and raised at 25°C. The resulting larval collection tubes would reach 3rd instar wandering stage around 4 days after hatching, at which stage plasmatocytes were extracted. PLASMATOCYTE ISOLATION AND CROSSLINKING FOR CHIP: For plasmatocyte extraction and chromatin fixation, 94h old 3rd instar wandering OrR larvae were collected from food tubes and washed in PBS. At the same time Schneider's medium FCS with 10 mM N-Acetyl-L-Cysteine was prepared in a well of a 6-well tissue culture plate (Sarstedt). Larvae were dried on paper towels and transferred to the tissue culture well with medium. Then they were ripped by holding the head with forceps (Dumont) and with another forceps ripping open the cuticula along the body of the larvae without disrupting the gut. 100 larvae were bled this way for each sample. The larval carcass was then removed and the plasmatocytes were allowed to attach for 10-15 minutes. Afterwards, the plasmatocytes were washed 4 times with PBS, then all supernatant was aspirated and plasmatocytes were fixed for 10 minutes at room temperature with 1,8% Formaldehyde in PBS. Afterwards, fixation was quenched for 3 minutes using 100 µl 2M glycine. Then cells were scraped of the dish using a cell scraper and transferred to 1,5 ml tube on ice. After a 5 minute incubation on ice, plasmatocytes were centrifuged (3000g, 4°C, 5 minutes), then washed 2x with PBS-Tx (PBS + 0,1% Triton X-100 w/v), each time spinning the cells down (3000g, 4°C, 5 minutes). After the final wash, as much supernatant as possible was removed from the pellet, without losing it, then the tube was snap frozen in liquid nitrogen and stored at -80°C for the ChIP.CHIP PROTOCOL: For ChIP a number of tubes corresponding to the appropriate number of fixed larval plasmatocytes (see Table 8) was thawed on ice. All tubes were then resuspended in ice cold PBS-Tx (PBS + 0,1% Triton X-100 w/v) and pooled. If several different ChIPs were performed in one run, they derive from a common cell pool. If replicates of a ChIP were performed in one run, these samples derive from separate cell pools. Pooled fixed plasmatocytes were centrifuged down (4500g, 4°C, 10 minutes) and all supernatant was drained. Cells were then resuspended in RIPA buffer supplemented with protease inhibitor cocktail, PhosSTOP and 20 mM sodium butyrate to a final concentration of 3 larval equivalents of plasmatocytes per µl. Plasmatocytes were then dissociated using a syringe with a G26 needle by passing the cell suspension through it 10 times and afterwards incubated for 30 minutes on ice. The chromatin suspension was next split into prechilled Bioruptor microtubes at 100 µl per tube and then sonicated in a Bioruptor Pico using 30s on 30s off cycles at 4°C. Sonication was performed first for 3 cycles and then 2 times for 4 cycles with in-between vortexing of tubes. Sonicated chromatin samples were then spun down to remove unsheared debris (16000g, 4°C, 10 minutes) and the supernatant was pooled into a fresh DNA LoBind tube. Samples were then split into chilled tubes according to the required plasmatocyte counts (see Table 8) holding a small amount back as input. Volumes were adjusted to 100 µl using RIPA and antibodies were added at appropriate concentrations (see Table 1). The chromatin antibody mixture was then incubated on a rotating wheel at 4°C overnight. The next day a 1:1 mixture of protein A and protein G Dynabeads was washed 3 times using a DynaMag-2. Then the beads were resuspended in a small volume and added to the overnight samples to make a final amount corresponding to 5 µl of each protein A and G beads. Bead-antibody-chromatin samples were incubated on a rotating wheel at 4°C for 4 hours. Beads were pulled down using a DynaMag-2 in a cold, resuspended in fresh RIPA and washed for 10 minutes on a rotating wheel. This washing was repeated 4 times with RIPA500, once with LiCl buffer and 2 times with TE buffer, for which washing was limited to 5 minutes. Finally, the beads were resuspended in 100 µl RIPA and the same was done for the untreated input sample. Then 1 µl of RNase (10 mg/ml) was added to each sample and they were incubated for 1h at 37°C. Then 4 µl 10% SDS and 3 µl Proteinase K (20 mg/ml) was added to each sample and they were incubated overnight at 65°C shaking at 1200 rpm. The next day the magnetic beads were removed using the DynaMag-2, and DNA was isolated from the supernatant using the Zymo ChIP DNA clean and concentrator kit according to the manual. The DNA was finally eluted in 30 µl ultra-pure water and stored at -80°C. ChIP-seq libraries were prepared from the final ChIP samples using the NEBNext Ultra II DNA kit and the NEBNext Multiplex Oligos for Illumina sets according to the manual with one minor adjustment: Size selection of the library was performed after PCR amplification. For this, after PCR amplification 27,5 µl of AMpure XP beads was added to each sample and incubated for 5 minutes. Then, the tubes were placed on a DynaMag-2 and the supernatant was moved to fresh tubes, while the beads were discarded. To the supernatant 22,5 µl AMpure XP beads was added, they were incubated for 5 minutes at room temperature, then pulled down using the DynaMag-2 and the supernatant was discarded. The beads were washed 2 times on the magnetic rack with 80% EtOH and then resuspended in 17 µl ultra-pure water. After incubating for 2 minutes at room temperature, the magnetic beads were pulled down and the supernatant containing the final libraries was moved to fresh tubes. The libraries were then stored at -20°C.