title: GSM5235941: WT2CpG2h ChIP-Seq; Mus musculus; ChIP-Seq
library_construction_protocol: For ChIP-Seq, BM-derived pDCs (CD3-CD19-CD11c+CD11blowB220+SiglecH+mPDCA1+) were FACS purified using FACS Aria III (BD). Cells were fixed with 1% formaldehyde for 15 min and quenched with 0.125 M glycine, and sent to Active Motif Services (Carlsbad, CA) to be processed for ChIP-Seq. In brief, chromatin was isolated by the addition of lysis buffer, followed by disruption with a Dounce homogenizer. Lysates were sonicated and the DNA sheared to an average length of 300-500 bp. Genomic DNA (Input) was prepared by treating aliquots of chromatin with RNase, proteinase K and heat for de-crosslinking, followed by ethanol precipitation. Pellets were resuspended and the resulting DNA was quantified on a NanoDrop spectrophotometer. Extrapolation to the original chromatin volume allowed quantitation of the total chromatin yield. An aliquot of chromatin (20 µg, spiked-in with 200 ng of Drosophila chromatin) was precleared with protein A agarose beads (Invitrogen). Genomic DNA regions of interest were isolated using 4 ug of antibody against BATF (CST, 8638BF). Antibody against H2Av (0.4 ug) was also present in the reaction to ensure efficient pull-down of the spike-in chromatin (Egan et al., 2016). Complexes were washed, eluted from the beads with SDS buffer, and subjected to RNase and proteinase K treatment. Crosslinks were reversed by incubation overnight at 65 °C, and ChIP DNA was purified by phenol-chloroform extraction and ethanol precipitation. Quantitative PCR (QPCR) reactions were carried out in triplicate on specific genomic regions using SYBR Green Supermix (Bio-Rad).  The resulting signals were normalized for primer efficiency by carrying out QPCR for each primer pair using Input DNA.