title: Illumina HiSeq 2500 sinlge-end Sequencing of KAS21009673 (KAE21103090)
library_construction_protocol: The cells were washed twice with PBS and incubated with 1% formaldehyde for 10 min at room temperature RT. The crosslinking was quenched with 125 mM glycine for 5 min at RT, and the cells were harvested with cold PBS and suspended in SDS lysis buffer 50 mM Tris-HCl, pH 8.0, 1% SDS, 10 mM EDTA. Following, the suspended chromatin was sheared using an ultrasonicator Covaris S220 and incubated overnight with the relavant antibodies against anti-CTCF Millipore; 07-729, anti-H3K27ac Abcam; ab4729, and Protein A, G sepharose GE Healthcare; 17-1279-03 and 17-0618-05 at 4degree Celsius with agitation. The immune complexes were washed sequentially with low-salt wash buffer 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, high-salt wash buffer 20 mM Tris-HCl, pH 8.0, 250 mM LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, and LiCl wash buffer, and finally washed twice with TE buffer. The washed immune complexes were eluted with elution buffer 1% SDS, 0.1 M NaHCO3 and de-crosslinked overnight at 68degree Celsius. The immunoprecipitated DNA was treated with proteinase K and RNase A, and collected by phenol-chloroform-isoamyl alcohol precipitation. Libraries were prepared using an Accel-NGS 2S Plus DNA Library Kit SWIFT; 21024 according to the manufacturer's guidelines. The libraries were sequenced using an Illumina Hiseq2500 system with the single-end method 50 bp.