title: GSM7984586: BPA-2-ERa-Cut&TAG; Homo sapiens; OTHER
library_construction_protocol: CUT&Tag was performed following the Bench top CUT&Tag V.3 protocol (Kaya-Okur, Wu et al. 2019). Briefly, we used 100K nuclei isolated from E2, BPA, or vehicle treated MCF7 cells per reaction. 10 ul BioMag-Plus Concanavalin (Bangs Laboratories) were used per reaction to immobilize the nuclei. Then we added 1:50 mouse ERα (sc-8005, Santa Cruz), 1:25 rabbit FOXA1 (#53528, Cell Signaling Technology), 1:50 mouse H3K27ac (#MA5-23516, Invitrogen), or 1:25 rabbit MED1 (#51613, Cell Signaling Technology) antibody and incubated for 2 hours. After one hour of secondary antibody incubation 1.25 ul of CUTANA pAG-Tn5 adapter complex was used to load the enzyme to the antibody bound regions. One hour of tagmentation at 37°C was followed by DNA extraction using MinElute PCR Purification Kit (Qiagen). Extracted DNA was subjected to PCR amplification using unique primers sets (Nextera XT v2 Full set (N7-S5)). Number of PCR cycles were determined specifically to prevent overamplification. We amplified ERα, FOXA1, H3K27ac, or MED1 with 14, 15, 15 or 17 PCR cycles, respectively. Libraries were size selected using AMPure XP Beads (Beckman Coulter) with 1.3X ratio. We quantified and assessed the quality of the libraries with Qubit Flex and Tapestation and then sequenced the libraries with 50 bp paired-end reads on Illumina high output NovaSeq SP.