Sample information curated by ChIP-Atlas

Antigen

Antigen Class
Input control
Antigen
Input control

Cell type

Cell type Class
Neural
Cell type
Microglia
MeSH Description
The third type of glial cell, along with astrocytes and oligodendrocytes (which together form the macroglia). Microglia vary in appearance depending on developmental stage, functional state, and anatomical location; subtype terms include ramified, perivascular, ameboid, resting, and activated. Microglia clearly are capable of phagocytosis and play an important role in a wide spectrum of neuropathologies. They have also been suggested to act in several other roles including in secretion (e.g., of cytokines and neural growth factors), in immunological processing (e.g., antigen presentation), and in central nervous system development and remodeling.

Attributes by original data submitter

Sample

source_name
microglia from 9 month-old animals
genotype
APP23
treatment
equal mixture of DNA from all treatment groups
chip antibody
none (input)

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
CD11bhigh/CD45low microglia were sorted by Fluorescence-activated cell sorting on a BD FACS Aria. For microglia isolation for chromatin purification, 1 mM sodium butyrate, an inhibitor of histone deacetylases, was added to the dissection medium and FACS buffers, and after staining, microglia were fixed in 1 % PFA for 10 minutes at room temperature, followed by addition of glycine (final concentration: 125 mM) for 5 minutes and washing in HBSS. Microglia were then sorted into homogenization buffer (0.32 M sucrose, 5 mM CaCl2, 5 mM MgAc2, 50 mM HEPES, 0.1 mM EDTA, 1 mM DTT, 0.1% vol/vol Triton-X-100) and centrifuged at 950 g for 5 minutes at 4 °C. The pellet was resuspended in 100 μl Nelson buffer (50 mM Tris, 150 mM NaCl, 20 mM EDTA, 1% vol/vol Triton-X-100, 0.5% vol/vol NP-40) and frozen on dry ice. The cross-linked chromatin was sheared for 3x7 cycles (30 sec. On/Off) in a BioruptorPlus (Diagenode) to achieve an average fragment size of 350 bps. Proper shearing and chromatin concentration was validated by DNA isolation and quantification using a small amount of each sample individually. Samples were split in half and 1 µg of ChiP-graded antibody (H3K4me1: Abcam ab8895; H3K27ac: Abcam ab4729) was added respectively and incubated overnight at 4ºC. From each sample, 1% of the total volume was taken as input prior to antibody binding. Immunoprecipitation was done by incubating samples with 30 µl BSA-blocked protein A magnetic beads (Dynabeads, Invitrogen) for 1hr at 4ºC. After purifying the precipitated chromatin and isolating the DNA, DNA libraries were generated using the NEB Next Ultra DNA Library Prep Kit for Illumina and the NEB Q5 polymerase (both from New England Biolabs). Multiplexing of samples was done using 6 different index-primers from the Library Prep Kit. One sample from each condition (genotype and treatment) was pooled for that purpose to rule out amplification and sequencing biases within the final data. Input samples were pooled and processed accordingly. The ideal number of amplification cycles was estimated via RealTime PCR to avoid over-amplification. Accordingly, samples were amplified for 13-15 cycles and the DNA was isolated afterwards. Individual libraries were pooled whereby each pool represented one whole batch of samples for each condition and targeted histone modification and was set to a final DNA concentration of 2 nM before sequencing (50 bp) on a HiSeq 2000 (Illumina) according to the manufacturer's instructions.

Sequencing Platform

instrument_model
Illumina HiSeq 2000

mm10

Number of total reads
132767723
Reads aligned (%)
89.9
Duplicates removed (%)
53.6
Number of peaks
837 (qval < 1E-05)

mm9

Number of total reads
132767723
Reads aligned (%)
89.3
Duplicates removed (%)
53.3
Number of peaks
1013 (qval < 1E-05)

Base call quality data from DBCLS SRA