Antigen

Antigen Class

Unclassified

Antigen

Unclassified

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.

Sample

source_name

sorted_microglia

strain

C57BL6

culture protocol

N/A

time in culture

ex vivo

ligands in culture

N/A

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

For ChIP-Seq experiments, a cross-linking step with paraformaldehyde was performed immediately after staining but before microglia sorting. Briefly, a subset of human cells, and mouse cells to be used for ChIP-Seq, were washed once after staining, fixed at RT with 1% paraformaldehyde in PBS (Affymetrix 19943 1 LT) for 10 minutes, incubated for 5 min at RT with glycine to a final concentration of 0.125 M for PFA quenching, and washed twice with cold staining buffer. Microglia were then sorted as described above. After sorting, microglia were pelleted and snap-frozen in liquid nitrogen and stored at -80 C until chromatin immunoprecipitation. Chromatin immunoprecipitation for histone modification H3K4me2, H3K27ac and transcription factor PU.1 was performed as followed, using 500,000 to 2 million microglia per assay. First microglia were briefly thawed on ice and lysed by incubation in 1 ml of lysis buffer (0.5% IGEPAL CA-630, 10 mM HEPES pH 7.9, 85 mM KCl, 1 mM EDTA pH 8.0, in water) for 10 min on ice. Lysate were centrifuge at 800 x rcf for 5 min at 4°C, and pellets were resuspended in 200 l of sonication/immunoprecipitation buffer (10 mM Tris-HCl pH 7.5, 100 mM NaCl, 0.5 mM EGTA, 0.1% Deoxcycholate, 0.5% Sarkosyl, in water). Sonication was performed with a Bioruptor Standard Sonicator (Diagenode) and consisted of two rounds of 15 min each, alternating stages of 30 sec “sonication-on” with 60 sec “sonication-off”. Twenty-two microliters of 10% Triton-X were then added to samples (1% final concentration) on ice, and lysates were cleared by centrifugation for 5 min at 18,000 x rcf at 4°C. Two microliters of supernatant were then set aside for input library sequencing controls. Supernatants were then immunoprecipitated on a rotator for 2 h at 4°C with antibody pre-bound to 20 l Protein A Dynabeads (Life Technologies 10001D). The following antibodies were used: H3k4me2 (Millipore, 07030), 2.0 l from stock per sample; H3K27ac (Active Motif, 39135), 1.5 l from stock per sample; and PU.1 (Santa Cruz, sc-352), 3.0 g per sample. Immunoprecipitates were washed three times each on ice with ice-cold wash buffer I (150 mM NaCl, 1% Triton X-100, 0.1% SDS, 2 mM EDTA pH 8.0 in water), wash buffer III (10 mM Tris-HCl, 250 mM LiCl, 1% IGEPAL CA-630, 0.7% Deoxycholate, 1mM EDTA in water) and TET (10 mM Tris-HCl pH 7.5, 1 mM EDTA pH 8.0, 0.1% Tween-20, in water) and eluted with 1% SDS/TE at room temperature in a final volume of 100 l. Reverse-crosslinking was then performed on immunoprecipitates and saved input aliquots. First, 6.38 l of 5 M NaCl (final concentration 300 mM) was added to each samples immersed in 100 l SDS/TE. Crosslinking was then reversed by overnight incubation at 65°C in a hot air oven. Potentially contaminated RNA was then digested for 1 h at 37°C with 0.33 mg/ml RNase A, proteins digested for 1 h at 55°C with 0.5 mg/ml proteinase K, and DNA extracted using Sera-Mag SpeedBeads (Thermo Scientific, 6515205050250). Sequencing libraries were prepared from recovered DNA (ChIP) or generated cDNA (RNA) by blunting, A-tailing, adapter ligation as previously described (2) using barcoded adapters (NextFlex, Bioo Scientific). Prior final PCR amplification, RNA-Seq libraries were digested by 30 min incubation at 37 C with Uracil DNA Glycosylase (final concentration of 0.134 units per l of library volume; UDG, Enzymatics G5010L) to generate strand-specific libraries. Libraries were PCR-amplified for 12-15 cycles and size selected for fragments (200-325 bp for ChIP-Seq, 200-350 for RNA-Seq) by gel extraction (10% TBE gels, Life Technologies EC62752BOX). RNA-Seq and ChIP-seq librairies were single-end sequenced for 51 cycles on an Illumina HiSeq 4000 or NextSeq 2500 (Illumina, San Diego, CA) according to manufacturer’s instruction. Mice were euthanized with CO2 and quickly perfused intracardially with ice-cold DPBS (Mediatech 21-031CV). Whole brains were removed and gently mechanically homogenized on ice as described above. Cells were fractionated by Percoll gradient centrifugation, washed, FC blocked with CD16/32 antibody (1:50, eBioscience 14-0161, clone 93) on ice for 15 min, stained 1:100 with CD11b-APC (BioLegend 101212, clone M1/70) and CD45-Alexa Fluor 488 (BioLegend 103122, clone 30-F11). Microglia were then purified by cell sorting as specified above, with microglia defined as live-CD11b+CD45Low single cells. Typical yield was roughly 250,000 microglia per mouse brain.

Sequencing Platform

instrument_model

Illumina HiSeq 2500

mm10

Number of total reads

20072002

Reads aligned (%)

88.5

Duplicates removed (%)

28.1

Number of peaks

23091 (qval < 1E-05)

mm9

Number of total reads

20072002

Reads aligned (%)

88.5

Duplicates removed (%)

28.2

Number of peaks

23095 (qval < 1E-05)