GSM5069845: pi-staining-and-freezing: pi_stained_frozen 4np (F38F7); Mus musculus; OTHER
220 nm cryosections were collected in sucrose droplets and transferred to steel frame PEN membrane slides. Slides were washed in 1x PBS (3 times, 5 min each), then with water (3 times, 5 min each). Staining was performed with propidium iodide (10 µg/ml in PBS) for 10 min, followed by 2 washes with water (30 s each) and air dryed for 15 minutes. Nuclear profiles were laser microdissected into adhesive 8-strip LCM collection caps, with four profiles dissected into each cap. Whole genome amplification was performed using the Sigma WGA4 kit. 14.5 µl of lysis and fragmentation mastermix (13 µl H2O, 1.4 µl lysis and fragmentation buffer, 0.1 µl proteinase K) was added to each well of a 96 well plate, caps with microdissected material were used to close the wells, then the plate was inverted and centrifuged upside-down at 3000 xg for 2 min such that the fragmentation mastermix was collected in the cap. Plates were incubated upside-down for 4 h at 50 ∞C then inverted and centrifuged right-way-up at 3000 xg for 2 min to collect the extracted DNA in the bottom of the well. Samples were then heat inactivated at 99 ∞C for 4 min then cooled on ice for 2 min. Samples were then frozen and stored overnight at -20 ∞C. The next day, 4.32 µl library preparation mastermix (2.88 µl library preparation buffer, 1.44 µl library stabilisation solution) was added to each sample, incubated at 95 ∞C for 2 min, cooled on ice for 2 min then centrifuged at 3000 xg for 2 min. 1.4 µl library preparation enzyme was added to each tube, then samples were incubated at 16 ∞C for 20 min, then 24 ∞C for 20 min, 37 ∞C for 20 min and 75 ∞C for 5 min. Finally, 87.84 µl amplification master mix (10.8 µl amplification buffer, 69.84 µl H2O, 7.2 µl WGA polymerase) was added to each tube and samples were amplified by PCR (initial denaturation 95 ∞C for 3 min, then 25 cycles of denaturation 95 ∞C for 30 s and annealing/extension 65 ∞C for 5 min). WGA amplified DNA fragments larger than 500bp were purified using Ampure XP beads and prepared for sequencing using the Illumina Nextera kit, following the manufacturer's guidelines