Pippin prep with a 3 agarose gel cassette

Small RNA-seq libraries were constructed using the NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (New England Biolabs, Inc., Ipswich, MA). The libraries were pooled, size-selected for products that were 120–135 nucleotides in length using a Pippin Prep with a 3% agarose gel cassette (Sage Science, Beverly, Massachusetts), and run on a MiSeq instrument (Illumina, San Diego, California) at the UC San Diego UCSD Institute for Genomic Medicine (IGM) Genomics Core. Samples that produced adequate numbers of miRNA read counts were then rebalanced to produce similar numbers of miRNA reads and sequenced on a HiSeq 4000 instrument using 1 × 75 bp reads (Illumina, San Diego, California) at the UC San Diego IGM Genomics Core. The data were trimmed and mapped to GRCh38 using the exceRpt Small RNA-seq Pipeline Workflow implemented in the Genboree Workbench.¹²⁴ (link) Micro RNAs were filtered such that at least five reads in at least two samples were retained, resulting in 1035 pass-filter miRNAs. Differential expression was carried out in DESeq2 (v.1.26.0)¹²² (link) requiring an adjusted p-value < 0.05 and log₂ FoldChange > 1 considered differentially expressed unless otherwise noted. As with the long RNA-seq, data from all lines created for this manuscript were compared to previously published naive and primed lines listed in (Table S5).

exRNA was isolated from each serum sample using the Plasma/Serum Circulating and Exosomal RNA Purification Kit (Slurry Format) (Norgen Biotek Corp., Ontario, Canada). Quality control of isolated RNA was performed using the Agilent RNA 6000 Pico Kit (Agilent, Santa Clara, CA). Small RNA-seq libraries were constructed using the NEB-Next Multiplex Small RNA Library Prep Set for Illumina (New England Biolabs Inc., Ipswich, MA). The libraries were then cleaned using the DNA Clean & Concentrator-5 Kit (Zymo Research, Irvine, CA), and quality control of the libraries was performed using the Agilent High Sensitivity DNA Kit (Agilent, Santa Clara, CA). Equal volumes of the libraries were pooled, size-selected for products that were 120 to 135 base pairs in length using a Pippin Prep with a 3% agarose gel cassette (Sage Science, Beverly, MA), and run on a MiSeq instrument at the UC San Diego Institute for Genomic Medicine (IGM) Genomics Core using the MiSeq Nano Reagent Kit (Illumina, San Diego, CA). Samples that produced adequate numbers of miRNA read counts were then rebalanced to produce similar numbers of miRNA reads and sequenced on a HiSeq 4000 instrument to produce 1 × 75 bp reads (Illumina, San Diego, CA) at the UC San Diego IGM Genomics Core.