Ribozero gold kit

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)).

Mosquito total RNA was extracted from the homogenizated per the manufacturer’s instructions by Quick-RNA miniprep kit (Zymo Research, Irvine, CA, United States). Each treatment was conducted in four replicates (n = 5 mosquitoes per replicate). The Ribo-Zero Gold Kit (Human/Mouse/Rat) was used to deplete ribosomal RNA (New England Biolabs, Ipswich, MA, United States). A total of 100 ng of depleted RNA per sample was sent for sequencing to The Genome Sequencing Laboratory at The University of Kansas (Lawrence, KS, United States), where the sample QC, libraries preparation, and were sequenced as 150 pb single reads in the Illumina HiSeq [NextSeq Mid-Output (MO)].