ChIP-Atlas: SRX7988728

Antigen

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.

library_strategy: ChIP-Seq
library_source: GENOMIC
library_selection: ChIP
library_construction_protocol: Microglia were fixed by 1% PFA immediately after staining and then subjected to sorting. Sorted microglia were then snap-frozen in liquid nitrogen and stored at −80°C until use. ChIP-seq libraries were constructed using ChIPmentation (Schmidl et al., 2015). In brief, approximately 200,000–300,000 sorted microglia were lysed and sonicated. Lysate was incubated with antibodies targeting PU.1 (3 µL) or H3K4me3 (2 μg) overnight at 4°C. After overnight incubation, 10 µL pre-blocked beads was added to each sample and incubated at 4°C for 2 h. Protein-bound beads were then washed and tagmented on beads with Tn5 at 37°C for 5 min. Then, samples were reverse-crosslinked with proteinase K (NEB) overnight. DNA was purified using a Qiagen MinElute Kit (28004). Purified DNA was subsequently amplified by PCR for 17–20 cycles, and the final libraries were size-selected (100–600 bp) by Ampure XP beads (Beckman Coulter). Pooled libraries were single-end sequenced on a NextSeq 500.