Bioanalyzer eukaryotic total rna nano assay

Nucleic acids were extracted by homogenizing the same half of placenta and brain tissue using a TissueLyser II (Qiagen) followed by the AllPrep DNA/RNA/miRNA Universal Kit (Qiagen) according to the manufacturer’s instructions. For the low-pass WGBS libraries, DNA was sonicated to ~350 bp using a E220 focused-ultrasonicator (Covaris) and bisulfite converted using the EZ DNA Methylation-Lightning Kit (Zymo Research) according to the manufacturer’s instructions. Libraries were prepared via the Accel-NGS Methyl-Seq DNA Library Kit (Swift Biosciences) with the Methyl-Seq Combinatorial Dual Indexing Kit (Swift Biosciences) according to the manufacturer’s instructions. The pool of 88 libraries was sequenced on all 4 lanes of an NovaSeq 6000 S4 flow cell (Illumina) for 150 bp paired end reads, which yielded ~65 million unique aligned reads (~6X genome cytosine coverage) for each sample. For the RNA-seq libraires, RNA integrity (RIN > 7) was confirmed using a Bioanalyzer Eukaryotic Total RNA Nano Assay (Agilent). Libraries were prepared with the KAPA mRNA HyperPrep kit (Roche) and NEXTFLEX Unique Dual Index Barcodes (PerkinElmer). The pool of 88 libraries was sequenced on 1 lane of a NovaSeq 6000 S4 flow cell (Illumina) for 150 bp paired end reads, which yielded approximately 25 million uniquely mapped reads for each sample.