title: GSM8189771: NSC, TF, DNA, rep1; Homo sapiens; OTHER
library_construction_protocol: Briefly, 10 ng of ChIP DNA were end-repaired, dA tailed and adaptor ligated using NEBNext ChIP-seq library preparation kit (E6240, NEB), according to manufacturer's instructions. Adaptor ligated DNA was eluted into 32 µL of 0.1xTE, 25 µL were used for ChIP-seq library preparation and 5 µl were processed for ChIP-STARR-seq plasmid libraries preparation. Briefly, 5 µL of purified adaptor ligated DNA for each replicate were separately amplified with primers 147 STARRseq libr FW and 148 STARRseq libr RV (Arnold et al., 2013), and used in a Gibson assembly reaction with an AgeI-SalI digested STARR-seq plasmid and a home-made Gibson reaction buffer (100mM Tris-HCl, 10mM MgCl2, 0.2 mM dNTP, 0.5U Phusion DNA polymerase (NEB), 0.16U 50 T5 exonuclease (NEB)). Gibson reactions for the ChIP replicates were pooled together, purified by ethanol precipitation and used for electroporation into electrocompetent MegaX DH10b E. coli bacteria (C640003, Invitrogen), according to manufacturer's instructions. A total of 5 electroporation reactions per library were performed with 2 µl of DNA each. After recovery, bacteria from the 5 reactions were pooled together and 100 µL of a 1:100 and 1:10000 dilution was plated on Ampicillin containing Agar plates to enable estimation of the number of transformants. The remaining 5 mL of bacteria culture were incubated in 1 L of LB-media supplemented with Ampicillin over-night, and the plasmid DNA was isolated using a Maxiprep kit (Macherey-Nagel) according to manufacturer's instructions.