anti-E2A antibody (V-18, Santa Cruz Biotechnology, Lot G0814)
molecule subtype
genomic DNA + crosslinked, bound protein
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Thymii were harvested from mice, and thymocytes were stained with appropriate antibodies for sorting. Following sort, Cells were fixed for 15 min in PBS containing 1.5 mM EGS(ethylene glycol-bis(succinic acid N-hydroxysuccinimide ester)), followed by incubation for 15 min with 1% formaldehyde. Formaldehyde was quenched by incubation for 10 min with 0.2 M glycine, and then washed twice with PBS and pellets were frozen in liquid nitrogen. Crosslinked cells were lysed, washed and sonicated for 22 cycles using a Biorupter Plus sonicator. Lysates were cleared with dilution buffer, and incubated overnight at 4°C with magnetic beads bound with 15 μg of anti-E2A antibody (V-18, Santa Cruz Biotechnology, Lot G0814) or 3 μl anti-H3K4me2 antibody (Millipore, 07-030, Lot 2430486). Beads were washed multiple times with appropriate buffers. Bound complexes were then eluted from beads by incubation for 30 min at 65 °C with shaking in elution buffer. Crosslinks were reversed by incubation overnight at 65°C. RNA and proteins were digested, followed by DNA purification using a ChIP DNA Clean and Concentrator kit (Zymoresearch). Libraries were prepared with the NEBNext primer set which involved applying ChIP DNA to end repair, A-tailing, adapter ligation, PCR amplification. They were then cleaned and size selected by AMPure beads (Agencourt)