Abi fast sybr green master mix

Total RNA was prepared from cells using RNeasy Plus Mini Kit (QIAGEN) following manufacturer's protocol. RNA concentration was measured using NanoDrop (Wilmington, DE, USA). Reverse transcription was performed using iScript DNA Synthesis Kit (Bio-Rad, Hercules, CA, USA) and qRT-PCR reactions were performed using ABI Fast SYBR Green Master Mix (Life Technologies, Grand Island, NY, USA) as previously described [36 (link)].

ChIP-qPCR was used to validate target genes derived from the ATAC-seq analysis. Three independent clonal isolates from LNCaP cells stably over-expressing HIST1H1A or control vector were used for ChIP assays as previously described [45 (link)]. Briefly, 1% formaldehyde was used to fixed cells, followed by cell lysis. Cell lysates were pre-cleared with Protein G Sepharose beads (GE Healthcare), then incubated with HIST1H1A (HPA043753, Sigma Life Science) or IgG (12-370, Millipore), and Protein G Sepharose beads were added for overnight incubation at 4° C. NaCl was used for reverse cross-linking, and DNA was extracted using Qiaquick PCR purification kit (Qiagen). The DNA product was used for ChIP-qPCR analysis, and samples were amplified in duplicates using ABI Fast SYBR Green Master Mix (Life Technologies, Grand Island, NY USA). Student’s T-tests was used to calculate statistical significance, and data are presented as mean ± SD with P < 0.05 being considered significant.

ChIP assays were performed as previously described [38 (link)]. Briefly, after pre-clearing with Protein G Sepharose beads (GE Healthcare, Piscataway, NJ, USA), antibodies targeting endogenous NDN (N-20, Santa Cruz, Santa Cruz, CA, USA), HA (anti-HA, Roche) or IgG (12-370, Millipore, Billerica, MA, USA) was added to the sonicated cell lysates and incubated at 4°C for 2 hours. Protein G Sepharose beads were then added and incubated overnight. The following day, immunocomplexes were washed twice with lysis buffer, once with high salt buffer (50 mM HEPES-KOH pH 7.5, 500 mM NaCl, 1 mM EDTA, 0.1% Triton X-100, 0.1% sodium deoxycholate), twice with LiCl buffer (10 mM Tris-HCl pH 8.0, 0.25 M LiCl, 0.5% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA) and once with TE buffer, followed by elution in TE buffer containing 1% SDS. Samples were incubated at 65°C overnight for reverse-crosslinking, and treated with RNAse A and proteinase K the next day. ChIP DNA was purified using QIAquick PCR Purification Kit (QIAGEN) according to manufacturer's protocol. The DNA obtained was analyzed either by high-throughput sequencing or qPCR. All ChIP-qPCR reactions were performed in duplicate using ABI Fast SYBR Green Master Mix (Life Technologies) mentioned above.