Ikaros (Ik-C, rabbit antibody (kind gift of S.Smale))
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Cells were fixed for 15 minutes in PBS 1.5mM Ethylene glycol-bis(succinic acid N-hydroxysuccinimide ester) (EGS), followed by 15 minutes with 1% formaldehyde. Formaldehyde was quenched for 10 minutes with 0.2M glycine. Cells were washed 2 times with PBS and pellets were frozen in liquid nitrogen. 30µl Protein G Dynabeads (Life Technologies) were blocked with 0.5% BSA (w/v) in PBS. Magnetic beads were bound with 6µg of indicated antibody. Crosslinked cells were lysed in lysis buffer 1 (50mM HEPES pH7.5, 140mM NaCl, 1mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% TritonX-100) and washed with lysis buffer 2 (10mM Tris-HCl pH8.0, 200mM NaCl, 1mM EDTA, 0.5mM EGTA). Cells were resuspended and sonicated in lysis buffer 3 (10mM Tris-HCl pH8.0, 100mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.1% Na-Deoxycholate, 0.5% N-lauroylsarcosine) for 10 cycles at 10 sec each on ice (20W) with 50 s on ice between cycles. Lysates were cleared by centrifugation and Triton X-100 was added at a final concentration of 1%. Lysates were then incubated overnight at 4°C with the previously prepared magnetic beads. Beads were washed once with RIPA (50mM Tris-HCl pH8.0, 150mM NaCl, 0.1% SDS, 0.1% Na-Deoxycholate, 1% Triton X-100, 1mM EDTA), once with RIPA 500 (50mM Tris-HCl pH8.0, 500mM NaCl, 0.1% SDS, 0.1% Na-Deoxycholate, 1% Triton X-100, 1mM EDTA), once with LiCl wash (10mM Tris-HCl pH8.0, 250mM LiCl, 0.5% NP-40, 0.5% Na-deoxycholate, 1mM EDTA) and finally twice with TE (10mM Tris pH8.0, 1mM EDTA). Bound complexes were eluted from the beads in elution buffer (10mM Tris-HCl pH8.0, 0.5% SDS, 300mM NaCl, 5mM EDTA) for 30 min at 65 °C with shaking. Crosslinks were reversed overnight at 65°C. RNA and protein were digested in the supernatant using RNase A and Proteinase K and DNA was purified using ChIP DNA clean and concentrator columns (Zymoresearch). Libraries were prepared according to Illumina's instructions. Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (minus exo) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation, ChIP DNA was PCR-amplified with Illumina primers with the respective indexes for 18 cycles and library fragments (ChIP) (insert plus adaptor and PCR primer sequences) were size- selected from a 8% polyacrylamide gel.