FB(OE)-CIRH1A mESC (constructed from BirA J1 mES cells)
cell type
embryonic stem cells
treatment
none
chip antibody
FLAG (Sigma-Aldrich, F3168)
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). Briefly, cells were crosslinked on plate with 3% FMA for 10 min, then quenched by 1/20 volume of 2.5 M glycine. after two times of ice cold PBS wash, cell were harvested by trypsin digestion, and lysis by 1 x nuclei lysis buffer. 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, the targeted DNA was eluted by elution buffer with protease K at 65℃. Libraries were prepared by NEBNext Ultra II DNA library preparation kit according to the standard protocols provided by the manufacturer.