Tissue tearor homogenizer

Tumors were sliced into thin sections manually with a sharp surgical blade. Sections were then incubated in RPMI media containing 10% FBS, L-glutamine, sodium pyruvate, penicillin/streptomycin, HEPES, nonessential amino acids, and beta-mercaptoethanol on 24-well polycarbonate Transwell inserts with a 0.4 μm pore size (Corning) and maintained in 5% CO₂ and atmospheric oxygen levels [18 (link)]. Tissues were incubated with viral peptides at 10 μg/mL or in equal volume of DMSO for 9 h. Tissues with poor viability after culture were excluded. Tumor sections were stored in RNAlater (ThermoFisher) at 4 degrees overnight, then stored at −80 until further processing. For RNA isolation, tissue was thawed on ice in 1 mL TRIZOL (Invitrogen), then homogenized with a Tissue Tearor homogenizer, BioSpec. RNA was then isolated following the TRIZOL recommended protocol. Resulting RNA was then further purified using Qiagen RNA Cleanup Kit. Peptides used in human studies: CLGGLLTMV (EBV_CLG), GLCTLVAML (EBV_GLC), NLVPMVATV (CMV_NLV), GILGFVFTL (IAV_GIL).

Tumor or parametrial fat pads were dissected and immediately placed in RNAlater Solution (Ambion, Life Technologies) and then stored at 4°C overnight. Tumor tissue was transferred to TRIzol Reagent (Ambion, Life Technologies), homogenized using the Tissue Tearor homogenizer (Biospec Products, Inc., Bartlesville, OK), and RNA was then isolated following the manufacturer’s protocol. Parametrial fat pad RNA was isolated using the RNeasy Lipid Tissue Mini Kit (Qiagen, Promega, Madison, WI) according to the manufacturer’s protocol. RNA concentrations were determined using a BioTek Synergy 2 microplate reader. cDNA was created from each RNA sample using the Maxima cDNA kit (Thermo Fisher). Transcripts were amplified using the KiCqStart SYBR Green qPCR ReadyMix, iQ (Sigma Life Science, Darmstadt, Germany) following the manufacturer’s protocol, and detected using a CFX96 Real-Time PCR Detection System (Bio-Rad). Each sample was analyzed in triplicate and each primer set was tested by a melting curve. Relative expression of each experimental transcript was normalized to mRNA expression of the ribosomal protein L32 or β-actin.

For tumor samples resected from UPS patients at the Hospital of the University of Pennsylvania, approximately 100 mg of tissue was minced into 1–2 mm pieces and incubated in 1% formaldehyde for 15 minutes. Formaldehyde was quenched with glycine at 0.125 M. Fixed tissue was homogenized for 60 seconds with a Tissue Tearor Homogenizer (Biospec) at 30,000 RPM. Homogenized tissue was washed with ice-cold PBS with 1X HALT protease inhibitor. For cell-line ChIP-RX, samples were fixed for 10 minutes in 1% formaldehyde quenched with glycine and washed with PBS as above. 5e6 S2 cells (Drosophila Melanogaster) were added to each sample of 2.5e7 for ChIP-RX normalization in downstream analysis.