GSM6605577
The purification of viable, cell-specific nuclei from brain tissue from Tam-induced Aldh1l1-cre/ERT2+; NuTRAP+ mice was achieved by combining two previously published protocols, with modifications. For each sample, two retinae were homogenized in 1 ml ice-cold nuclei EZ lysis buffer (#NUC-101, Millipore Sigma) supplemented with 1X Halt protease inhibitor cocktail (Thermo Fisher Scientific) and 1 mM DTT using a glass dounce tissue grinder set (20 times with pestle A and 20 times with pestle B). Undissociated tissue, largely composed of blood vessels, was removed by centrifugation at 200 x g for 1.5 min at 4°C, and the supernatant containing the nuclear material filtered through a 30 µm strainer and centrifuged at 500 x g for 5 min at 4°C. The resulting nuclear pellet was resuspended in nuclei lysis EZ buffer, incubated on ice for 5 min, washed by centrifugation, and resuspended in 200 µl nuclei EZ storage buffer by gentle trituration with a micropipette. From the total resuspended pellet volume, 10% was reserved as input nuclei fraction and the rest was diluted with 1.6 ml nuclei purification buffer (NPB: 20 mM HEPES, 40 mM NaCl, 90 mM KCl, 2 mM EDTA, 0.5 mM EGTA, 1X Halt protease inhibitor cocktail, 1mM DTT), and subjected to the INTACT protocol. Briefly, 30 µl of resuspended M-280 Streptavidin Dynabeads (#11205, Thermo Fisher Scientific) were added into a fresh 2 ml microcentrifuge tube and washed with 1 ml of NPB using a DynaMag-2 magnet (#12321; Thermo Fisher Scientific) for a total of three washes (1 min incubation/each). The washed beads were reconstituted to their initial volume (30 µl) with NPB and gently mixed with the nuclear suspension. The mixture of nuclei and magnetic beads was incubated at 4°C for 40 min under gentle rotation settings to allow the affinity binding of streptavidin beads to the cell-specific, biotinylated nuclei. After incubation, the streptavidin-bound nuclei were magnetically separated with the DynaMag-2 magnet for a period of 3 min. The nuclei bound to the beads were washed in the magnet for three washes (1 min/each), resuspended in 30 µl of NPB, and reserved as the positive nuclei fraction. From the nuclear fraction [input and positive (enriched in biotinylated nuclei)], an aliquot was mixed with equal volume of DAPI counterstain and used for confocal microscopy visualization. The AllPrep DNA/RNA kit Micro (#80284, Qiagen, Germantown, MD) was used to extract gDNA and nuclear RNA from each sample. gDNA and nuclear RNA were quantified with a Nanodrop One spectrophotometer (Thermo Fisher Scientific) and its quality assessed by genomic DNA D1000 (#5067-5582) and HSRNA ScreenTape, respectively, with a 4150 TapeStation analyzer. For each input and positive INTACT-isolated sample 400 ng of gDNA was brought up to 50 µl volume with 1X low-EDTA TE buffer and sheared with a Covaris E220 sonicator (Covaris, Inc., Woburn, MA) to an average 200 base pair size using the following settings: intensity of 5, duty cycle of 10%, 200 cycles per burst, 2 cycles of 60 seconds, at 7°C. The size of sheared products was confirmed by capillary electrophoresis (DNA D1000, Agilent).  gDNA fragments were cleaned by an Agencourt bead-based purification protocol, after which gDNA was quantified (Qubit dsDNA, Thermo Fisher Scientific). Two aliquots of 62.2 ng DNA fragments were prepared in a 12 µl volume to which 1 µl of spike-in control DNA (0.08 ng/ul) with known levels of specific mC, hmC, and fC at individual sites was added.  End repair, ligation of methylated adaptors (#L2V11DR-BC 1-96 adaptor plate, NuGEN, Tecan Genomics, Inc., Redwood City, CA) and final repair were performed according to manufacturer's instructions (Ovation Ultralow Methyl-Seq Library System, NuGEN).