Nextseq 500 genome analyzer

For transcriptomic studies, paired end sequencing was performed using Illumina HiSeq 2500 (Illumina, San Diego, CA, USA) with a 108 bp read length at the Center for Biotechnology and Genomics Core Facility at Texas Tech University to study gene expression. For miRNA profiling, Illumina small RNA protocol (Illumina, Inc., San Diego, CA, USA) was used for libraries preparation and sequencing, then the Illumina Genome Analyzer NextSeq 500 (Illumina, Inc., San Diego, CA, USA) was used to perform sequencing at Department of Molecular and Cell Biology, Baylor College of Medicine, Houston.

ChIP assays were carried out on D341, D283 and MSCs cultures of approximately 2–5 million cells per sample and per epitope, following the procedures described previously (46 (link), 47 (link)). In brief, chromatin from formaldehyde-fixed cells was fragmented to a size range of 200–700 bases with a Branson 250 sonifier. Solubilized chromatin was immunoprecipitated with antibodies overnight at 4C. Antibody-chromatin complexes were pulled down with protein G-Dynabeads (Life Technologies), washed, and then eluted. After crosslink reversal, RNase A, and proteinase K treatment, immunoprecipitated DNA was extracted with AMP Pure beads (Beckman Coulter). ChIP DNA was quantified with Qubit. 2-5 ng ChIP DNA samples were used to prepare sequencing libraries, and ChIP DNA and input controls were sequenced with the Nextseq 500 Illumina genome analyzer. For primary tissue preparations, 10-30 mg of tumor was first cut on dry ice and chopped on ice with a razor blade. Samples were then resuspended in 1 mL cold PBS and fixed 15 minutes at room temperature by adding formaldehyde to a final concentration of 1%. Glycine was added for 5 minutes at room temperature. Samples were first washed in 1 mL cold PBS and resuspended in 1 mL cold PBS before manual homogenization with a syringe and processing as described above.