title: GSM9499506: E6.5 Epi,Sp1 Ctr,Ser2p,ChIP,rep1; Mus musculus; ChIP-Seq
library_construction_protocol: ChIP-seq was performed with minor adjustments to the STAR ChIP-seq protocol. Samples were lysed in a buffer containing 0.5% Tween-20, 0.5% NP-40, and 0.1% SDS, and gently pipetted to release genomic DNA. The mixture was then digested with MNase (Sigma) at 37°C for 5 minutes. Primary antibodies—anti-Pol II RBP1 (BioLegend), anti-Phospho-POLR2A CTD-S2 (Abclonal), anti-SP1(Proteintech), anti-H3K4me3 (Millipore) or anti-H3K27me3 (Abclonal)—were added, followed by incubation at 4°C overnight. Protein A Dynabeads (Thermo Fisher Scientific) were added to the solution and incubated for 3-4 hours. Beads were washed five times with RIPA buffer containing 0.1% protease inhibitor and once with LiCl wash buffer. Elution of DNA fragments was achieved with proteinase K treatment. The library was constructed using NEB Next® UltraTM II DNA Library Prep Kit according to the operation instruction.