Illumina HiSeq 3000 paired end sequencing; RNA-seq of Drosophila plasmatocyte mosaics which lack PRC2/H3K27me3
RNA-seq of Drosophila plasmatocyte mosaics which lack PRC2/H3K27me3
FLY RAISING Flies were raised and maintained on standard cornmeal food at 25°C or room temperature. 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.  CROSSES FOR MUTANT MOSAICS Crosses for the final larvae were left to lay eggs for 4h using 20 female and 10 male flies. The resulting larvae were then heat shocked twice, once in the 1st instar stadium (24h-48h after egg laying) and once in the 2nd instar stadium (48h-72h after egg laying). Then 3rd instar wandering non-tb female larvae were collected for further processing.  PLASMATOCYTE ISOLATION AND FACS SORTING For FACS sorting of plasmatocytes 94h old 3rd instar wandering larvae were collected (infered genotypes: (MARCM;; Su(z)12^4,FRT2A/tubGal80,FRT2A), (MARCM;; E(z)^731,FRT2A/tubGal80,FRT2A), (hsFlp;HisC,FRT40A/ubiGFP,FRT40A;HisRescue)). These larvae were washed in PBS, dried on paper towels and transferred to Nunclon Sphera ultra-low attachment dishes filled with 0,5 ml Schneider's medium with FCS with 10 mM N-Acetyl-L-Cysteine. Larvae were then bled 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. Carcasses were then removed and the cell suspension was cell stained using 70 µm cell FlowMi cell strainers. DAPI was added to a final concentration of 1 µg/ml to mark dead cells.  Sorting of cells for RNA isolation was performed in the Flow Cytometry Core Facility (DRFZ), using the BD FACSAria ROWLF, sorting for GFP using a 488nm laser for excitation and a 530/30 filter for emission and sorting for DAPI negative cells using a 405nm laser for excitation and a 450/40 filter for emission. The resulting cells were collected, spun down (2000g, 4°C, 5 minutes) and lysed either in 500 µl TRIzol (H3 wt and H3 K27R mosaics) or 94.5 µl RNAdvance lysis buffer (Su(z)12 and E(z) mosaics). TRIzol samples were stored at -80°C, RNAdvance samples were isolated immediately. RNA ISOLATION BY TRIZOL Frozen TRIzol samples were thawed at room temperature, adjuster to 900 µl with TRIzol and moved to fresh prespun phase lock heavy tube. 250 µl chloroform was added to each sample, they were mixed thoroughly and centrifuged (12000g, room temperature, 15 minutes). The upper aqueous phase was then moved to a fresh DNA LoBind tube, making sure not to disrupt the interphase or phenol phase, and mixed with 550 µl isopropanol and 1 µl glycogen (20 mg/ml, RNAse free). Samples were mixed by inverting and incubated for 30 minutes at in the freezer at -20°C. Afterwards samples were centrifuged (16ooog, 4°C, 10 minutes) and the supernatant was removed carefully without disrupting the pellet. The pellet was resuspended in 100 µl ultra-pure water with 300 mM sodium acetate and 1 µl glycogen (20 mg/ml, RNAse free). Next, 300 µl EtOH was added and the sample was incubated for 20 minutes at -20°C, then centrifuged (16000g, 4°C, 10 minutes) and the supernatant was discarded. The pellet was washed 2 times by adding 1 ml of 70% EtOH (prepared with ultra-pure water), each time spinning down the pellet (16000g, 4°C, 3 minutes). Afterwards all supernatant was drained and the pellet was dried until no liquid was visible. The pellet was then resuspended in 15 µl of ultra-pure water and stored at -80°C.   RNA ISOLATION BY RNADVANCE RNA was isolated from freshly lysed plasmatocytes samples using the RNAdvance cell v2 kit following manufacturer's instructions using a DynaMag-2 magnetic rack. DNase digestion was performed as suggested in the manual using Ambion DNase. Resulting RNA samples were eluted in 20 µl ultra-pure water and stored at -80°C. Libraries for FACSed plasmatocytes from HisC mosaics were generated by the Max Planck Genome Centre (Cologne). RNA-seq libraries from Su(z)12/E(z) mosaics using the NEBNext Ultra II Directional RNA Library Prep with Sample Purification Beads, the NEBNext Poly(A) mRNA Magnetic Isolation Module and the NEBNext Multiplex Oligos for Illumina sets according to manufacturer's instruction. Adaptor concentrations were adjusted according to manual instructs.