title: GSM9052174: Y1_PIMQ_lowH3K4me1_Rep1; Mus musculus; ChIP-Seq
library_construction_protocol: Using 50,000 FACS-purified EpdSCs per target histone mark for each biological replicate, with two biological replicates per condition and 2-3 mice pooled per biological replicate. Briefly, purified samples were split in two (25,000 cells each), then lysed for 20 minutes on ice in the presence of either low (60 units) or high (120 units) concentration of micrococcal nuclease (NEB). Sample/MNase concentration-specific T7 adapters were then ligated to each sample. Following ligation, samples for each MNase concentration were pooled together, then split evenly for overnight incubation with ChIP antibodies in 4°C (H2A.Z: rabbit, AbCam; H3K4me1: rabbit, Cell Signaling; H3K27ac: rabbit, Active Motif). Antibody-bound DNA was isolated the following day through 4h incubation with protein G beads in 4°C, after which samples were washed twice with ice-cold RIPA, then once each with high salt RIPA, LiCL wash buffer, and TE buffer. Eluted DNA was then cleaned and subjected to overnight in vitro transcription in 4°C. Resulting RNA was isolated and reverse transcribed Library PCR was performed with sample-specific barcodes for pooled sequencing. Finally, libraries were cleaned, pooled, then sequenced for 50bp paired-end reads on an Illumina NextSeq P2000.