GSM4432214: APP_Con_2_H3K4me3; Mus musculus; ChIP-Seq
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.