Truseq standard mrna sample preparation kit

Library preparation and RNA sequencing were performed by HELIXIO with TruSeq® Standard mRNA sample Preparation kit (Illumina) and NextSeq500 platform (Illumina) applying paired-end sequencing (2 × 75 bp). We then proceeded to the analysis of the sequencing data using ASE-TIGAR after quality check by FastQC, trimming and pairing of the resulting reads using custom Perl scripts. First, we derived mRNA reference sequences from new reference genomes and merged them in one FASTA file, then mapped the clean reads on the mRNA reference with Bowtie2 [96 (link)]. We then run ASE-TIGAR with SAM files produced by mapping and the mRNA reference (http://nagasakilab.csml.org/ase-tigar/) [20 ].
Raw sequencing data is available on NCBI database under the SRA accession number SRP154565 (https://www.ncbi.nlm.nih.gov/sra/SRP154565).

In order to evaluate the effects of maternal malnutrition on the offspring VP miRNAs expression profile, the total RNA of the VP samples from the CTR group (n = 5) and the GLLP (n = 4) group were extracted using Trizol following the datasheet guidelines (Thermo Fisher, Waltham, MA, USA). The purified RNA was stored at −80 °C in DNase/RNase-free distilled water. The Nano Vue Plus spectrophotometer (GE Healthcare, Chicago, IL, USA) was used to verify the quality of RNA extraction and quantification, and the RNA Integrity Number (RIN) was determined by analyzing microfluidic ribosomal RNAs using the Agilent 2100 Bioanalyzer system (Santa Clara, CA, USA) [79 (link)]. Only samples with a RIN of 8 or above were used. The RNA was sequenced in a HiSeq2500 platform (Illumina, San Diego, CA, USA). For rRNA depletion, an aliquot of total unfractionated RNA was provided for a library building, and Ribo-Zero sequencing was employed during library preparation. The TruSeq Standard mRNA Sample Preparation Kit (Illumina) was used to purify messenger RNA and build libraries from total RNA, according to the manufacturer’s instructions. Small non-coding (Snc) RNA libraries were prepared by TruSeq Small RNA Library Prep Kit (Illumina, CA, USA) following the manufacturer’s specifications.

72 postmortem PFC samples were selected from the 72 individuals used for lipidome measurements, with square-root- transformed ages uniformly distributed along the whole lifespan. Of them, 19 individuals were measured previously [30 (link)]. Sample weights are 20–30 mg. RNA integrity numbers (RIN) of 62 out of the 72 samples were not lower than six. For the other ten samples, although their RINs were lower than six or unmeasurable, their 28S/18S rRNA ratios were around two, supporting that these samples were suitable for RNA-seq measurement. More detailed information of human samples is provided in Table S1. The poly(A) + RNA enriched cDNA library was constructed using the Illumina TruSeq® standard mRNA sample preparation kit. The RNA fraction was sequenced on the Illumina HiSeq 4000 platform in pair-ended mode, each mate in length of 150 nt. The sample order was randomized prior to RNA extraction and sequencing. The person performing RNA extraction, cDNA library construction, and RNA sequencing were unaware of sample information, including age and ethnicity.