Nexflex dna barcodes 24

Libraries were prepared using 150 ng of total RNA from bone marrow NK cells and splenic NK subpopulations and KAPA Stranded RNA-Seq Library Preparation Kit (KK8400, KAPA Biosystems, Wilmington, MA), and each library was indexed using NEXflex DNA Barcodes-24 (NOVA-514103, BIOO Scientific, Austin, TX). Barcoded PCR products were purified on 2% E-Gel, 250–400 bp fragments were purified, quantified on Qbit (Invitrogen), mixed, and sequenced on an Illumina HiSeq 2500 or HiSeq 3000 platform (Illumina, San Diego, CA).

Splenic NK cells from 10 WT and 13 Stat5 DKI mice were purified (Miltenyi Biotec Inc.), expanded in vitro with 20 ng ml⁻¹ of human recombinant IL-15 (R&D Systems or BioLegend) for 10 days, with fresh rhIL-15 being added every two days, yielding 50–80 million of nearly 100% pure CD3⁻CD122⁺NK1.1⁺ cells. The cells were washed three times with medium, rested in complete RPMI-1640 medium without IL-15 for 2 h, not treated or treated with 40 ng ml⁻¹ of IL-15 for 1 h, and cross-linked with 1% formaldehyde (methanol-free, Pierce, Rockford, IL) at room temperature for 10 min. After sonication, fragmented chromatin equivalent to 10 million cells were immunoprecipitated with control rabbit IgG (sc-3888, Santa Cruz Biotechnology, Dallas TX) or anti-STAT5B (AF1584, R&D Systems) and Magna ChIP Protein A + G Magnetic Beads (16–663, Millipore, Billerica MA). ChIP-Seq DNA libraries were prepared using KAPA LTP Library Preparation Kit (KK8232, Kapa Biosystems, Wilmington, MA) and NEXflex DNA Barcodes-24 (NOVA-514103, BIOO Scientific, Austin, TX), and the libraries were then sequenced on an Illumina HiSeq 3000 platform.