Thruplex fd prep kit

Using a 2-mm² core needle, approximately three cores (TF) were extracted from areas circled on each slide. Frozen cores were pulverized using the Covaris CryoPrep system. Tissue was then fixed using 1% formaldehyde with methanol for 18 min at room temperature, and fixation was quenched with 2M glycine. Chromatin was sheared to 300–500 bp in size using the Covaris E220 ultrasonicator. The resulting chromatin was incubated overnight with 6 µg of antibody—to AR (E2724, Spring Biosciences), HOXB13 (sc-66923, Santa Cruz Biotechnology) or FOXA1 (ab23738, Abcam)—bound to protein A and protein G beads (Life Technologies). A fraction of the sample was not exposed to antibody and was used as control (input). Samples underwent cross-linking reversal and were treated with RNase and proteinase K, and DNA was extracted. Concentrations of ChIP DNA were quantified by Qubit fluorometer (Life Technologies). DNA sequencing libraries were prepared using the ThruPLEX-FD Prep kit (Rubicon Genomics). Libraries were sequenced using 75-bp reads on the Illumina platform at the Dana-Farber Cancer Institute.

H3K4me3 ChIP was performed in T47D cells with 2.5×10⁶ cells/IP, as previously described (Carroll et al., 2005 (link)). In brief, cells were treated as indicated. Cells were crosslinked, lysed in SDS lysis buffer, and sonicated (Covaris). H3K4me3 antibody (3ug) was added to sonicated lysates per o/n IP. Protein A beads (Novex) were incubated with IP for 6h. Chromatin was eluted and reverse crosslinked o/n and DNA was recovered with RNAse A incubation. Glycogen and Proteinase K were added and incubated at 62°C. DNA was purified and eluted in Low TE. KDM5A and HA ChIPs were performed using the Sarkosyl method (Lee et al., 2006 (link)) with 2.5×10⁸ cells/ChIP and KDM5A #1416 antibody (10ug) (Dr. William Kaelin) or HA ab9110 (5ug) (abcam), respectively.
For samples that were Illumina sequenced, ChIP eluate was purified (Ampure) and indices added (ThruPLEX-FD Prep kit, Rubicon Genomics). Samples were then size selected (Pippin Prep, Sage Science) and purified (Ampure). DNA quality and quantity was measured using a Fragment Bioanalyzer and samples submitted to the Center for Functional Cancer Epigenetics for sequencing on a Next-Seq Illumina platform.

H3K27Ac ChIP in 22RV1 cells was performed as previously described (Pomerantz et al., 2015 (link)). Ten million cells were fixed using 1% formaldehyde (Thermo fisher, Waltham, MA) for 10 minutes at 37°C. Chromatin was sheared in ice cold lysis buffer (50mM Tris, 10mM EDTA, 1% SDS with protease inhibitor) to 300–500 base pairs using the Covaris E210 sonicator. The sample was incubated with 1 µg H3K27Ac antibody (Diagenode, C15410196, Denville, NJ) coupled with protein A and protein G beads (Life Technologies, Carlsbad, CA) at 4°C overnight. The chromatin was washed with RIPA washing buffer (0.05M HEPES pH 7.6, 1 mM EDTA, 0.7% Na Deoxycholate, 1% NP-40, 0.5M LiCl). After decrosslinking, IP DNA as well as its input were extracted using Qiagen Qiaquick columns, and sequencing libraries prepared using the ThruPLEX-FD Prep Kit (Rubicon Genomics, Ann Arbor, MI). Libraries were sequenced using 75-base pair single reads on the Illumina platform at the Dana-Farber Cancer Institute.

Radical prostatectomy tissue was prepared and ChIP-Seq performed as described previously (20 (link)) with 6-ug antibodies to AR and H3K27Ac antibody. DNA sequencing libraries were prepared using the ThruPLEX-FD Prep Kit (Rubicon Genomics, Ann Arbor, MI). Libraries were sequenced using 50-base pair reads on the Illumina platform (Illumina, San Diego, CA) at Dana-Farber Cancer Institute. Related data analysis for ChIP-Seq and peak calling followed the same protocol as reported. The quantitation of signals on the 1-MB region of enhancer of RRM2 was performed from 4 normal prostate tissue samples and 4 tumor samples. The data are available from GEO (GSE118845).
ChIP-qPCR was performed using the ChIP-IT High Sensitivity Kit (Active Motif, #53040) with ChIP-grade H3K4me3, FOXM1 antibodies and native IgG, according to the manufacturer’s instructions. DNA was analyzed via qPCR. All ChIP experiments were completed with at least two biological replicates.
For luciferase reporter assays, siRNAs were transfected in cells for 24 hours and 500 ng of RRM2 promoter reporters (GeneCopoeia) containing 0kb or 3kb promoter sequence of human RRM2 were cotransfected with Lipofectamine 2000 (Invitrogen). At 24 hours post-transfection, cells were collected for luciferase assays according to Promega’s instructions.

ChIP and ChIP-reChIP were performed as described (Jehle et al., 2014 (link); Xu et al., 2012 (link)). For ChIP-seq, sequencing libraries were generated according to manufacturer's instructions (ThruPLEX-FD Prep Kit, Rubicon Genomics) and then sequenced on the Illumina Hiseq-2000 platform.

ChIP experiments were conducted as described previously (Wang et al., 2009 (link)) and were done in triplicates. Chromatin from approximately 1 × 10⁷ fixed cells was sonicated to a size range of 200-300 bp. Solubilized chromatin was subjected to immunoprecipitation with the ER antibody, Ab10 (Lab Vision corporation) and SC-543 (Santa cruz), HA antibody (Ab9110, Abcam), H3K27Ac antibody (C15410196, Diagenode) or FOXA1 (ab5089,Abcam) bound to protein A and protein G beads (Life Technologies). A fraction of the sample was not exposed to antibody to be used as control (input). The samples were reversed crosslinked, treated with proteinase K, and DNA was extracted. DNA sequencing libraries were prepared using the ThruPLEX-FD Prep Kit (Rubicon Genomics). Libraries were sequenced using 50 bp reads on the Illumina Nextseq500 at the Dana-Farber Cancer Institute.