A liposarcoma containing myxomatous tissue. (Dorland, 27th ed)
Attributes by original data submitter
Sample
source_name
cancer cell line
cell line
MLPS-402-94
experimental condition
genotype: FUS-DDIT3 fusion shDDIT3
chip antibody
SMARCC1
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
ChIP-seq protocol Cells were harvested following 72-hr exposure to specified lentivirus and 4 day selection with 2 μg/ml of puromycin. ChIP experiments were performed per standard protocols (Millipore, Billerica, MA) with minor modifications. Briefly, cells were cross-linked for 10 min with 1% formaldehyde at 37 °C. This reaction was subsequently quenched with 125 mM glycine for 5 min and 10 million fixed cells were used per ChIP experiment. Chromatin from fixed cells was fragmented by sonication with a Covaris E220 and the solubilized chromatin was incubated with the indicated antibody listed overnight at 4°C. Antibody-chromatin complexes were pulled down by incubation with Protein G-Dynabeads (Thermo Scientific) for 2 hours at 4°C, washed, and eluted. The samples then underwent crosslink reversal, RNase A (Roche) treatment, and proteinase K (Thermo Scientific) treatment before the captured DNA was extracted with AMPure XP beads (Beckman Coulter). ChIP-seq libraries were prepared with Illumina's NEBNext Ultra II DNA library Prep Kit using standard protocols. ChIP-seq was sequenced on Illumina Next-seq 500 using 75 bp single-end sequencing parameters. ATAC-seq protocol Cells were harvested following 72-hr exposure to lentivirus and 4 days of selection with 2 μg/ml of puromycin. ATAC-seq experiments were performed as previously described. Samples for ATAC-seq prepared using the Nextera DNA sample Prep Kit (Illumina). Briefly, 50K cells were incubated in hypotonic buffer and lysis buffer, then were resuspended in transposase reaction mixture for 30 min at 37°C with gentle shaking followed by DNA purification and 12 cycles of amplification. ATAC-seq samples were sequenced on Next-seq 500 (Illumina) using 37 bp pair-end sequencing parameters. RNA-seq protocol Cells were harvested following 72-hr exposure to the specified lentivirus and either 4 day (day 7 post-transduction) or 11 day (day 14 post-transduction) selection with 2 μg/ml of puromycin for RNA-seq experiments. All RNA was collected using the RNeasy Mini Kit (Qiagen). RNA-seq libraries were prepared using the Nebnext Poly(A) mRNA Magnetic Isolation Module and the Nebnext Ultra II Directional RNA Library Prep Kit for Illumina using standard protocols. RNA was sequenced on Illumina Next-seq 500 (Illumina) using 75 bp single-end sequencing parameters. Snap-frozen tumor tissue samples were obtained from the MD Anderson Institutional Tissue Bank (ITB) using an IRB-approved protocol. Frozen samples were embedded in O.C.T. (Tissue-Tek), sectioned in a Cryostat (Leica CM1680 UV) and underwent hematoxylin and eosin staining for pathologist review to confirm the presence of viable tumor tissue. Tissues were then excised from O.C.T. using scalpel and forceps and 30 mg samples were placed in RNAlater-ICE (Invitrogen) pre-chilled to -80°C. Samples were thawed overnight at -20°C, then disrupted using a TissueLyser LT (Qiagen) for 5 minutes at 50 Hz. RNA was isolated using the RNeasy Mini Kit (Qiagen), following the manufacturer protocol with on-column DNase I (Qiagen) digestion. Cut & Run protocol Cells were harvested following 24-hr exposure to DMSO or 10uM of CMP12 (generous gift of Jun Qi) followed by 24-hr exposure to DMSO plus base media or StemPro Adipogenesis media (ThermoFisher) or 10uM CMP12 plus base media or StemPro Adipogenesis media. CUT&RUN experiments were performed according to established protocols with minor modifications. Briefly, 500K cells per sample were incubated with Concanavalin A-coated beads (Polysciences) for 10 min and then permeabilized with Triton X-100. Cell-bead complexes were then incubated with the indicated antibody for 2 hr at 4°C. Cell-bead complexes were washed and then incubated with pA-MNase for 1 hr at 4°C. Cell-bead complexes were washed and then cooled to ~0°C. pA-MNase was activated by the addition of calcium chloride and incubated for 30 min at ~0°C. The cleavage reaction was quenched with a solution containing spike-in DNA and RNAase A. Cleaved DNA fragments released into solution were then collected and treated with sodium dodecyl sulfate (SDS) and proteinase K (Thermo Scientific). DNA was then isolated using phenol-chloroform. CUT&RUN libraries were prepared with Illumina's NEBNext Ultra II DNA library Prep Kit with modifications that aim to preserve short fragments. ChIP-seq libraries were prepared with Illumina's NEBNext Ultra II DNA library Prep Kit using standard protocols. ChIP-seq was sequenced on Illumina Next-seq 500 using 75 bp single-end sequencing parameters. CUT&RUN libraries were prepared with Illumina's NEBNext Ultra II DNA library Prep Kit with modifications that aim to preserve short fragments. ATAC-seq samples were sequenced on Next-seq 500 (Illumina) using 37 bp pair-end sequencing parameters. RNA-seq libraries were prepared using the Nebnext Poly(A) mRNA Magnetic Isolation Module and the Nebnext Ultra II Directional RNA Library Prep Kit for Illumina using standard protocols. RNA was sequenced on Illumina Next-seq 500 (Illumina) using 75 bp single-end sequencing parameters.