Transcriptome Profiling of Pediatric PBMC

Total RNA was obtained from six PBMC samples from 1-y-old females with a range in ΔHAZ scores. ERCC spike-in RNA (Illumina) was added to aliquots of total RNA per instructions of the manufacturer, ribosomal RNA was then depleted using the RiboZero gold kit and libraries were constructed using the NEBNext Ultradirectional RNA Lib Prep Kit (Cat #E74205) and NEBNext Multiplex Oligos (Cat#E73355). The 75-bp paired-end reads were obtained from the NextSeq500 instrument described above. Raw reads were assessed by FASTQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and mapped to the hg19 reference genome using HISAT2 (55 (link)). RNAs were then assembled and quantified using StringTie (56 (link)) and differential analysis was performed on the resulting transcripts count table using DESeq2 (51 (link)).