Nebnext chip seq library prep kit

For ChIP followed by qPCR, 2–5 million cells were used as starting material; for ChIP-seq, 20–50 million cells were used. Cells were trypsinized, washed, and cross-linked as described previously (Sen et al. 2008 (link)). Lysis with a modified RIPA buffer (1% NP40/Igepal, 0.5% Sodium Deoxycholate, 0.1% SDS, 100 uM EDTA in PBS), nuclei were isolated and overnight pulldown was performed with an antibody to MITF (Sigma HPA003259), H3K27ac (Abcam ab4729), H3K4me1 (Abcam ab8895), or Rabbit IgG control (Abcam ab37415). Staph A cells were used for pulldown and washes were performed as described previously (Sen et al. 2008 (link)). Isolated DNA was subject to qPCR with primers in Supplemental Table 6. High-throughput ChIP-sequencing libraries were prepared with a modified version of NEBNext ChIP-seq library prep kit (NEB) using AMPure beads (Agencourt) for purification. Barcodes were introduced and these libraries were sequenced with 1 × 50-bp reads on the Illumina HiSeq platform.

Freshly isolated AM (1 × 10⁶) were cultured and expanded in complete RPMI supplemented with 25 ng/mL murine GM-CSF (Peprotech) for 2 weeks. Chromatin immunoprecipitation (ChIP) was performed using the SimpleChIP (Cell Signaling Technology) following the manufacture's manual. Briefly, 20 × 10⁶ AM were treated with formaldehyde at a final concentration of 1% to cross-link DNA-protein complexes. Cells were lysed and DNA-protein complexes were sheared by micrococcal nuclease, followed by precipitation with nonspecific rabbit anti-IgG or Anti-VDR (D2K6W) (Cell Signaling Technology. Eluted and purified ChIP DNA was used to prepare the DNA-sequencing libraries with the NEBnext ChIPSeq library Prep kit (NEB) for Next-Generation Sequencing (Illumina). Raw sequencing data was deposited in GEO with accession ID: GSE124725. ChIP-seq were analyzed with the software package HOMER [58 (link)].