in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with
10% FBS, 100 U/mL penicillin and 100 mg/mL streptomycin. For the
transfection experiments, the cells were subjected to viral infection
with recombinant adenoviruses encoding TP53BP2 (Ad-TP53BP2),
adenoviruses encoding DNMT1 (Ad-DNMT1), adenoviruses encoding G9a
(Ad-E2F1), and adenoviruses encoding G9a (Ad-G9a), and adenoviruses
encoding green fluorescent protein were used as a negative control
(Ad-NC). Following a 4-hour incubation, the culture medium was replaced
with regular RPMI-1640 supplemented with 7% FBS, and the cells were
then exposed to hypoxic conditions at 37°C with 5% CO2for 48 h. Additionally, these two cell lines were transfected with
lentiviruses carrying sh-TP53BP2, sh-Beclin-1, sh-DNMT1, sh-E2F1, or
sh-G9a, and sh-NC via Lipofectamine 2000 (Thermo Fisher Scientific, USA)
following the manufacturer’s protocol. After 6 h of incubation, the
cells were cultured in 7% fetal bovine serum under hypoxic conditions
at 37°C with 5% CO2 for 48 h.
RNA-sequencing (RNA-seq) assay
Total RNA was extracted from placental tissues to construct cDNA
libraries. For the small RNA cDNA library, the complete RNA was first
ligated with both an RNA 3′ adapter and a 5′ adapter. Subsequently,
reverse transcription primers were used to convert the ligated RNAs into
cDNAs. The resulting cDNAs were then amplified through polymerase chain
reaction (PCR) and purified via gel electrophoresis. The quality of the
cDNAs was assessed via an Agilent 2100 chip (Agilent, Santa Clara, USA).
For the analysis of the RNA-seq library, the total RNA was purified to
eliminate rRNA with the Ribo-Zero™ rRNA Removal Kit (Epicenter-Illumina,
Madison, USA), followed by fragmentation of the RNA. The fragmented RNA
was then converted into first-strand cDNA via a TruSeq® Stranded Kit
(Epicenter-Illumina, Madison, USA). Double-stranded cDNA was generated
via DNA polymerase I and RNase H. The 3′ ends of the double-stranded
cDNA were adenylated and then ligated with adapters. PCR amplification
and purification were carried out to construct the cDNA library. The
libraries were sequenced via the Illumina HiSeq 2500 platform for total
RNA sequencing, which employs a 90-bp paired-end sequencing strategy,
whereas small RNA sequencing was performed via the Illumina HiSeq X Ten
platform.