48 hour in vitro culture with IL-2 (growth protocol 1)
chip antibody
input
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
ChIP-seq: Cells were chemically cross-linked by 1% formaldehyde. Lysates were made by sonication (10,000,000 cells/sample) and protein-DNA complexes isolated using the indicated antibodies. ChIP-seq: Single-end libraries were prepared for STAT5-bound fragments, along with un-precipitated input controls, using the NEBNext ChIP-Seq Library Prep kit for Illumina (New England Biolabs). Briefly, DNA fragments were end-repaired to generate phosphorylated blunt ends. These were then treated with Taq polymerase and dATP to generate 3-prime 'A' base overhangs for ligation to adapters containing complementary 'T' base overhangs. After ligation, DNA was PCR amplified for 15 cycles with indexed primers and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel, and quantitated by Qubit (Invitrogen). ChIP-seq: Indexed libraries were multiplexed and sequenced for 50 single read cycles on Illumina HiSeq 2000 or 2500 instruments according to the manufacturer's instructions. RNA-seq: RNA was extracted from 50-500K cells by phenol-chloroform method with GlycoBlue as co-precipitant (7 μg per sample; Life Technologies). RNA-seq: Single-end libraries were prepared from 0.1-0.5 μg of total RNA using the TruSeq RNA Sample Preparation Kit V2 (Illumina) RNA-seq: Indexed libraries were multiplexed and sequenced for 50 single read cycles on Illumina HiSeq 2000 or 2500 instruments according to the manufacturer's instructions.