title: GSM4158659: C10-OXBS; Mus musculus; Bisulfite-Seq
library_construction_protocol: The purification of viable, cell-specific nuclei from brain tissue from Tam-induced Aldh1l1-cre/ERT2+; NuTRAP+ and Cx3cr1-cre/ERT2+; NuTRAP+ mice was achieved by combining two previously published protocols, with modifications.10,43 For each mouse, a hemisected half-brain was rinsed in ice-cold 1X PBS, minced into small pieces and homogenized in 4 ml ice-cold nuclei EZ lysis buffer (#NUC-101, Millipore Sigma) supplemented with 1X HaltTM protease inhibitor cocktail (ThermoFisher Scientific) using a glass dounce tissue grinder set (#D9063; Millipore Sigma: 20 times with pestle A and 20 times with pestle B).43 Undissociated tissue, largely composed of blood vessels, was removed by centrifugation at 200 x g for 1.5 min at 4°C,44 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 300 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 HaltTM protease inhibitor cocktail), and subjected to the INTACT protocol.10 Briefly, 30 µl of resuspended M-280 Streptavidin Dynabeads (#11205, ThermoFisher Scientific) were added into a fresh 2 ml microcentrifuge tube and washed with 1ml of NPB using a DynaMag-2 magnet (#12321; ThermoFisher 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 and the unbound nuclei collected in a fresh 2 ml microcentrifuge tube, centrifuged at 4°C (1,000 x g, 3 min), resuspended in 100 µl of NPB and reserved as the negative nuclei fraction. 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 each nuclear fraction (input, negative, and positive), a 3 µl aliquot was mixed with equal volume of DAPI counterstain and used for confocal microscopy visualization and calculation of purity percentage (3-5 fields of view per sample). The AllPrep DNA/RNA kit Micro (#80284, Qiagen, Germantown, MD) was used to extract gDNA from each sample.10 gDNA was quantified with a Nanodrop 2000c spectrophotometer (Thermofisher Scientific) and its quality assessed by genomic DNA D1000 (#5067-5582) with a 2200 Tapestation analyzer (Agilent Technologies, Santa Clara, CA). For each input, negative, 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 (QubitTM dsDNA, Thermofisher Scientific). Two aliquots of 200 ng gDNA fragments were prepared in a 12 µl volume to which 1µl of spike-in control DNA (0.08 ng/ul) with known levels of specific mC, hmC, and fC at individual sites was added. Whole genome oxidative bisulfite (WGoxBS) libraries were constructed with NuGen Ovation Ultra-low Methyl Seq Library System with True-Methyl OXBS module.
attributes.tag: GEO Accession
attributes.value: GSM4158659