Comment[GEAAccession]	E-GEAD-665
MAGE-TAB Version	1.1
Investigation Title	CRISPR screening in human trophoblast stem cells reveals both shared and distinct aspects of human and mouse placental development
Experiment Description	The placenta serves as the interface between the mother and fetus, facilitating the exchange of gases and nutrients between their separate blood circulation systems. Trophoblasts in the placenta play a central role in this process. Our current understanding of mammalian trophoblast development relies largely on mouse models. However, given the diversification of mammalian placentas, findings from the mouse placenta cannot be readily extrapolated to other mammalian species, including humans. To fill this knowledge gap, we performed CRISPR knockout (KO) screening in human trophoblast stem cells (hTSCs). We targeted genes essential for mouse placental development and identified more than 100 genes as critical regulators in both human hTSCs and mouse placentas. Among them, we further characterized in detail two transcription factors, DLX3 and GCM1, and revealed their essential roles in hTSC differentiation. Moreover, a gene function-based comparison between human and mouse trophoblast subtypes suggests that their relationship may differ significantly from previous assumptions based on tissue localization or cellular function. Notably, our data reveal that hTSCs may not be analogous to mouse TSCs or the extraembryonic ectoderm (ExE) in which in vivo TSCs reside. Instead, hTSCs may be analogous to progenitor cells in the mouse ectoplacental cone and chorion. This finding is consistent with the absence of ExE-like structures during human placental development. Our data not only deepen our understanding of human trophoblast development but also facilitate cross-species comparison of mammalian placentas.
Experimental Design	case control design
Experimental Factor Name	cell_type	genetic_modification
Experimental Factor Type	cell_type	genetic_modification
Person Last Name	Kobayashi
Person First Name	Eri
Person Affiliation	Informative genetics, Tohoku University, School of Medice
Person Roles	submitter
Public Release Date	2023-12-26
Publication DOI	10.1073/pnas.2311372120.
Protocol Name	P-GEAD-2087	P-GEAD-2088	P-GEAD-2089	P-GEAD-2090	P-GEAD-2091	P-GEAD-2092
Protocol Type	sample collection protocol	nucleic acid extraction protocol	nucleic acid labeling protocol	nucleic acid hybridization to array protocol	array scanning and feature extraction protocol	normalization data transformation protocol
Protocol Description	hTSCs were maintained in hTSC medium [DMEM/F12 (FUJIFILM Wako #048-29785) supplemented with 1% knockout serum replacement (KSR) (Thermo Fisher Scientific #10828028), 0.5% penicillin-streptomycin (Thermo Fisher Scientific #15140122), 0.15% bovine serum albumin (BSA) (FUJIFILM Wako #017-22231), 1% ITS-X supplement (FUJIFILM Wako #094-06761), 200 uM L-ascorbic acid (FUJIFILM Wako #013-12061), 25 ng/ml EGF (FUJIFILM Wako #053-07871), 2 uM CHIR99021 (FUJIFILM Wako #038-23101), 5 uM A83-01 (FUJIFILM Wako #035-24113), 0.8 mM valproic acid (FUJIFILM Wako #227-01071), and 2.5 uM Y27632 (FUJIFILM Wako #257-00511)] at 37?C in 95% air and 5% CO2. When hTSCs reached sub-confluence, they were dissociated with TrypLE Express (Thermo Fisher Scientific #12604021) diluted with PBS at a 1:1 ratio for 10-15 min at 37?C. Dissociated cells were seeded in hTSC medium supplemented with 0.5 ug/ml iMatrix-511 (Nippi #892011). hTSCs were typically passaged every two days at a split ratio of 1:4 to 1:8. For CRISPR screening and gene KO, 10 ng/ml BMP4 (R&D Systems #314-BP) was added to the hTSC medium to ameliorate the toxicity of gene transfection and antibiotic selection. To induce EVT differentiation, hTSCs were cultured in EVT medium [DMEM/F12 supplemented with 0.5% penicillin-streptomycin, 0.15% BSA, 1% ITS-X supplement, 50 ng/ml NRG1 (Cell Signaling #5218SC or #26941), 7.5 uM A83-01, 2.5 uM Y-27632, and 4% KSR] supplemented with 2% Matrigel (Corning #354263) for three days. The culture medium was then replaced with EVT medium without NRG1. Fro ST diffirentiation, hTSCs were cultured on plates coated with 1 ug/ml Col IV (Corning #354233) using ST medium [DMEM/F12 supplemented with 0.5% penicillin-streptomycin, 0.15% BSA, 1% ITS-X supplement, 2.5 uM Y-27632, 2 uM forskolin, and 4% KSR].	Total RNA was extracted using RNeasy Mini Kit and RNase-Free DNase (QIAGEN #74536 and #79254). For ChIP-seq, Cells were fixed with 1% PFA for 10 min. After quenching and cell lysis, chromatin was sonicated. For HiChIP, Approximately 10 million cells were fixed in 1% PFA for 10 min and 3 mM disuccinimidyl glutarate for 40 min as previously described. Fixed NIH3T3 cells were added to the cross-linked cells as spike-in controls. The cell mixtures were lysed in HiC Lysis Buffer [10 mM Tris-HCl (pH 7.5), 10 mM NaCl, and 0.2% IGEPAL CA-630], and the isolated nuclei were resuspended in 0.5% sodium dodecyl sulfate (SDS) and permeabilized at 62?C for 10 min. Next, a 3.3-fold volume of 1.5% Triton X-100 was added and incubated at 37?C for 15 min. In situ digestion with DpnII (NEB #R0543) was performed at 37?C for 60 min, and the enzyme was heat-inactivated at 62?C for 20 min. Overhangs were biotin-labeled using DNA Polymerase I large (Klenow) Fragment (NEB #M0210) at 37?C for 60 min in the presence of biotin-14-dATP (Thermo Fisher Scientific #19524016), dTTP, dCTP, and dGTP. Proximity ends were ligated with 13.3 units/?l of T4 DNA ligase (NEB #M0202) at 23?C for 4 h. The nuclei were pelleted and treated with 0.5 units/?L of Exonuclease III (NEB #M0206S) for 5 min at 37?C, resuspended in Nuclear Lysis Buffer [50 mM Tris (pH7.5), 10 mM EDTA, and 1% SDS], and sonicated using Covaris M220 (M&S Instruments Inc.) to 300-700 bp length. Sonicated DNA was diluted 1:10 with ChIP Dilution Buffer [50 mM Tris (pH 7.5), 165 mM NaCl, 1.1% Triton X-100, and 0.01% SDS] and clarified by centrifugation. An anti-H3K4me3 antibody (Merck Millipore, Clone #CMA304) was added to the fragmented DNA at 1:500 dilution, and the mixture was rotated at 4 ?C for 60 min. The IP complex was captured using Dynabeads M280 anti-mouse (Life technologies #11201D), and the beads were washed four times with High-Salt Wash Buffer [20 mM Tris (pH 7.5), 500 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS, and 0.1% sodium deoxycholate] and twice with 10 mM Tris-HCl (pH7.5). The washed beads were suspended in Extraction Buffer [10 mM Tris (pH8.0), 350 mM NaCl, 0.1 mM EDTA, and 1% SDS] supplemented with 5% volume of proteinase K (Takara, #9034), and incubated at 55?C for 1 h and 67?C for 2 h to reverse the crosslinks. ChIPed DNA was purified using MinElute PCR Purification Kit (QIAGEN #28004). For biotin pull-down, 5 ?l of Dynabeads MyOne Streptavidin C1 beads (Life Technologies #65001) were suspended in 2x Biotin Binding Buffer [10 mM Tris-HCl (pH 7.5), 1 mM EDTA, and 2 M NaCl] and mixed with an equal volume of ChIPed DNA. After 50 min of rotation at room temperature, the beads were washed with Tween Wash Buffer [5 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, 1 M NaCl, 0.05% Tween-20] and NEB T4 Ligase Buffer (NEB # B0202), and resuspended in 10 mM Tris-HCl (pH 8.0).	nucleic acid library construction protocol: RNA-Seq libraries were constructed using NEBNext UltraII Directional RNA Library Prep Kit (NEB). ChIP-Seq libraries of TFs were constructed using NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB # E7645) with two custom adaptors: Adapter-T and Adapter-C. Adapter-T with a 3'-T overhang was prepared by annealing (5'-CTA CAC GAC GCT CTT CCG ATC TT-3') and (5'-AGA TCG GAA GAG CAC ACG TCT GAA-3'; 5' phosphorylated). Adapter-C with a 3'-C overhang was prepared by annealing (5'- CTA CAC GAC GCT CTT CCG ATC TC-3') and (5'-AGA TCG GAA GAG CAC ACG TCT GAA-3'; 5' phosphorylated). Adapter-C was added to increase the ligation efficiency. HiChIP libraries were constructed using KAPA Hyper Prep Kit (NIPPON Genetics #7962312001) and TruSeq-compatible duplex Y adapter (IDT). After PCR amplification, the amplicon was size-selected using Ampure XP (Beckman Coulter #A63880).	nucleic acid sequencing protocol: Libraries were sequenced on an Illumina NovaSeq 6000 platform with 150 bp paired-end reads (Rhelixa) or Illumina HiSeq2500 with single-end reads	dummy	Sequenced RNA-seq reads were trimmed for quality control using TrimGalore v0.6.7 and aligned to the reference genome (UCSC hg38) using STAR v2.7.10a with the RefSeq gene annotation. ChIP-Seq reads were trimmed for quality control using TrimGalore v0.6.7 and mapped to the reference genome (UCSC hg38) using Bowtie 2 v2.2.5. HiChIP reads were mapped to the reference genome (UCSC hg38) using the HiC-Pro pipeline with default settings, and data from replicates were merged. Significant long-range (>20 kb) chromatin interactions were calculated using FitHiChIP. H3K4me3 ChIP-Seq data from hTSCs, hTSC-derived STs, and hTSC-derived EVTs were used as inputs for FitHiChIP. Only interactions with at least one end overlapping the H3K4me3 ChIP-Seq peaks were retained.
SDRF File	E-GEAD-665.sdrf.txt
Comment[Number of channel]	single-channel
Comment[Array Design REF]	A-GEAD-11
Comment[AEExperimentType]	transcription profiling by array
Comment[NBDC]	The Data Access Committee of the National Bioscience Database Center (NBDC) approved that this personal data was made published according to the NBDC Guidelines for Human Data Sharing (https://humandbs.biosciencedbc.jp/en/guidelines/data-sharing-guidelines) as the NBDC Research ID hum0433 and the application ID J-DS000928-001.
Comment[BioProject]	PRJDB17215
Comment[Related study]	JGA:JGAS000659	NBDC:hum0433
Comment[Last Update Date]	2023-12-26