Long-read transcriptome assembly reveals vast transcriptional complexity in the placenta associated with metabolic and endocrine function

Sample Source: Subset of two mother-offspring cohort studies comprising N=72 term live births without known placentation pathologies (e.g., intrauterine growth restriction, pre-eclampsia). Fifty percent of samples were from pregnancies affected by gestational diabetes mellitus (GDM), categorized by 75g two-timepoint oral glucose tolerance test using WHO 1999 criteria. Sample Processing: Placental tissue was processed within 1 hour of delivery. Five villous biopsies from each placenta were rinsed with phosphate buffer saline (PBS), snap-frozen in liquid nitrogen, and stored at -80 degrees C prior to RNA extraction. Cohort Details: N=6 samples from the Growing Up in Singapore Toward Healthy Outcomes (GUSTO) birth cohort with matched Illumina short-read libraries from the same biological specimens; N=66 samples from an independent Singaporean birth cohort. RNA Extraction: GUSTO samples (N=6) were extracted from approximately 100 mg crushed tissue using phenol-chloroform and purified with Qiagen RNeasy Mini Kit. Independent cohort samples (N=66) were extracted from approximately 75 mg crushed tissue combined with 500 microliters Buffer ATL (Qiagen, Cat #939011) and 30 microliters Proteinase K (Qiagen QIASymphony DSP DNA Midi Kit) and incubated overnight in a shaking incubator. Sequencing: Samples were sequenced on PromethION platform (release 23.04.6, Oxford Nanopore) with 3x 24-plex multiplexing using PCR-cDNA library prep kit (SQK-PCB111.24) at the Genome Institute of Singapore following manufacturer's protocols. Median read length was approximately 1,000 bp.