ChIP-seq in human trophoblast stem cells

Trophoblasts are multifunctional cells in the placenta and essential for normal pregnancy. Although trophoblast dysfunction can cause pregnancy complications, their underlying mechanisms remain unclear, and effective treatments are limited, partly because of the scarcity of appropriate experimental models. We previously reported the derivation of human trophoblast stem cells (hTSCs) from 1st-trimester placentas and blastocysts, providing a powerful tool to investigate human trophoblast development and function. However, the difficulty in deriving hTSCs from late-gestation placentas has limited their use in pregnancy complication research. Here we report a technique to derive hTSCs from term placentas based on the transient expression of a p53 dominant negative mutant, SALL4, and shRNAs against cyclin-dependent kinase inhibitors. Using this technique, we derived hTSCs from placentas obtained from patients with preeclampsia and reproduced key features of the disease. hTSCs were transduced with pCS-3G-FLAG-SALL4 or an empty vector using lentivirus and treated with 200 ng/ml Dox. ChIP was performed using ChIP Reagents (#318-07131; Nippon Gene, Tokyo, Japan) and an anti-FLAG antibody (1:100; #F3165; Sigma-Aldrich, St. Louis, MO, USA). IP was performed in buffer containing 400 mM NaCl instead of 150 mM to reduce the background signal. ChIP-seq libraries were prepared using NEBNext Ultra II DNA Library Prep Kit for Illumina (#E7645; New England Biolabs, Ipswich, MA, USA) and sequenced on an Illumina NovaSeq 6000 platform. Reads were trimmed using TrimGalore and aligned to the reference genome (UCSC hg38) using Bowtie 2 v2.2.5. SALL4-binding sites were identified using MACS2 v2.2.7.2.