Illumina HiSeq 2500 paired end sequencing; Placental epitranscriptome-wide analysis of revealed the m6A modification level at 5' UTR related fetal growth
Placental epitranscriptome-wide analysis of revealed the m6A modification level at 5' UTR related fetal growth
The placenta was collected from pregnant women who received cesarean section with explanations and consent regarding participation in research and chorionic villi was collected within 15 minutes after delivery. RNA samples were chemically fragmented into 200-nucleotide-long fragments by 25-second incubation at 94 ℃ in Nature fragmentation buffer (10mM ZnCl2, 10mM Tris-HCl, pH 7). The fragmentation reaction was stopped with 0.5 M EDTA (pH 8.0) and then kept on the ice, and next fragmented RNA for immunoprecipitation was purified by RNeasy MinElute Cleanup Kit (QIAGEN) as instruction. Finally, the size of each sample was checked by Agilent 2100 Bioanalyzer (Agilent). The reverse transcription reaction of input and immunoprecipitated IP RNA samples was carried out with SuperScript Ⅲ (Invitrogen) as manufacturer instruction. Washed in PBS three times to remove maternal blood, the refined villi (about 1 mg) was put into TRIzol® (Life Technologies) and homogenized by a scissor within 15 min after placental delivery. Total RNA was extracted following TRIzol® instruction. Human embryonic kidney cells 293 (HEK293T) cell was incubated in DMEM (Gibco, Invitrogen) containing 4.5 g/L D-glucose and GlutaMAX™ supplemented with 10% FBS and penicillin/streptomycin at 37 ℃ in 5% CO2. Total RNA with RNeasy Mini Kit (QIAGEN) was extracted following the instruction. DNA was removed using an RNase-Free DNase Set (QIAGEN). Enrichment of poly(A) RNA from total RNA of chorionic villi and HEK293T cells was performed two rounds by Dynabeads® Oligo(dT)25 (invitrogen™). The quality of each total RNA and poly(A) RNA was checked by Agilent 2100 Bioanalyzer (Agilent). Strand-specific and 101 bp paired-end RNA-seq libraries were generated with SureSelect Strand Specific RNA (Agilent) following manufacturer's protocol.
Experimental Factor: immunoprecipitate
input DNA
Experimental Factor: clinical information
small for date with maternal preeclampsia