FB(OE)-SAFB mESC (constructed from BirA J1 mES cells)
cell type
embryonic stem cells
treatment
none
chip antibody
none
affinity purification
none
strain
129/Ola
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
ChIP was done following previously protocol with modifications (Shen, 2008). Cells were crosslinked by 2 mM DSP (dithiobis succinimidyl propionate) for 30 minutes, followed by a 10-minute 1% formaldehyde crosslinking. The crosslinked cells were first partially fragmented by 12 U/ml DNase I at 37 °C for 10 minutes, then sonicated at 25% amplitude for 30 seconds. The lysate were further sonicated into average DNA size 200-500 bp. spin at 14000rpm for 15 min, transferred the supernatant into a new tube and diluted by adding 5 × volume of ChIP dilution buffer. Added 5 μg FLAG antibody, end to end rotate overnight at 4℃. The target were further captured by adding 20 ul Protein A/G UltraLink Resin, further rotated for 4 hours at 4℃. After thoroughly washing, these samples were subjected to a second purification step using 30 μl M-280 Streptavidin Dynabeads (Invitrogen 11205D). The rest of the steps were performed as previously described (Shen et al., 2008) Libraries were prepared by NEBNext Ultra II DNA library preparation kit according to the standard protocols provided by the manufacturer.