Antigen

Antigen Class

Bisulfite-Seq

Antigen

Bisulfite-Seq

Cell type

Cell type Class

Neural

Cell type

Brain

MeSH Description

The part of CENTRAL NERVOUS SYSTEM that is contained within the skull (CRANIUM). Arising from the NEURAL TUBE, the embryonic brain is comprised of three major parts including PROSENCEPHALON (the forebrain); MESENCEPHALON (the midbrain); and RHOMBENCEPHALON (the hindbrain). The developed brain consists of CEREBRUM; CEREBELLUM; and other structures in the BRAIN STEM.

Sample

source_name

Brain

genotype

Aldh1l1-Cre/Ert2+; NuTRAP+

fraction

Negative

animal number

275

library type

Whole genome bisulfite

library prep kit

Nugen Ovation Ultra-Low Methyl Seq with True-Methyl OxBS module

Sequenced DNA Library

library_strategy

Bisulfite-Seq

library_source

GENOMIC

library_selection

RANDOM

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.

Sequencing Platform

instrument_model

Illumina NovaSeq 6000

mm10

Number of total reads

7633489

Reads aligned (%)

89.1

Coverage rate (×)

0.3

Number of hyper MRs

318986 (qval < 1E-05)

mm9

Number of total reads

7633489

Reads aligned (%)

90.2

Coverage rate (×)

0.3

Number of hyper MRs

318932 (qval < 1E-05)