GSM6161001: 17A031042-M; Homo sapiens; Bisulfite-Seq
Within 15 min after delivery, 5 ml of neonatal umbilical cord blood was collected using two PAXGenRNA sampling tubes (2.5 ml/tube), and collected samples were were stored at -80 ℃. The placental tissues were collected from both the maternal and fetal side. Specifically, using a clean scalpel, one full-thick placental tissue was incised 5 cm from the perimeter of the placenta (a size of about 1×1×2 cm, including maternal and fetal side). Then, the tissues were rinsed with phosphate buffer (PBS) or normal saline until no visible blood and stored at -80 ℃ in a cryopreservation tube. DNA or RNA was extracted after homogenizing the tissues together. RRBS and RNAseq library construction protocol -- Genomic DNA of placenta and umbilical cord blood specimens were extracted by using the TIANamp genomic DNA kit (Cat. No. DP304-02, TIANGEN Biotech CO. LTD., Beijing, China) per the manufacture's recommendations. 50-300 ng of purified genomic DNA was utilized to generate RRBS libraries as previously described (https://www.nature.com/articles/s41594-018-0046-4). Indexed RRBS libraries were pooled accordingly and sequenced in a NovaSeq 6000 sequencer (Illumina, USA) with 100-base paired-end reads. Similarly, total RNAs were isolated using a miRNeasy Mini kit (QIAGEN, Germany) according to the manufacture's recommendations. We used the Ribo-off rRNA depletion kit (Cat. No. N406-01, Vazyme Biotech Co. Ltd, Nanjing, China) to remove ribosomal RNA from total RNAs following the manufacturer's instructions. To generate RNA-seq libraries, 10-100 ng rRNA-depleted RNAs were utilized by using the VAHTS total RNA-seq (H/M/R) library prep kit for Illumina (Cat. No. NR603-01, Vazyme Biotech Co. Ltd, Nanjing, China) per the manufacturer's instructions. We sequenced the RNA-seq libraries in the NovoSeq 6000 sequencer with 100-base paired-end reads. The sequencing data volume of a single sample is around 8 gigabases (Gb) and the average sequencing depth of the target region of the enzyme digestion fragment is > 30×, the coverage proportion of CpG region is > 40%, and the total data volume is 100 Gb. Similarly, transcriptome profile for each placenta and umbilical cord blood sample was also conducted. VAHTSTM Total RNA-seq (H/M/R) Library Prep Kit for Illumina® based on Ribo-zero method was used to constructed transcriptome RNA library and high-throughput sequencing was performed subsequently. 10 Gb volume of transcriptome data for each sample was necessary.