title: small RNA-seq of human: adult T4 PNK treated total RNA from plasma anticoagulated with EDTA
design: Total extracellular RNA (exRNA) was isolated from 450 l serum or platelet-poor plasma using a customized RNA isolation protocol that we developed to quantitatively inhibit nuclease activity (Max et al, PNAS, 2018, doi: 10.1073/pnas.1714397115). The plasma samples were anticoagulated with EDTA, acid-dextrose citrate (ACD), or unfractionated heparin. Half of the isolated total exRNA from serum or plasma was treated with T4 polynucleotide kinase (PNK, NEB, cat# M0201S) for 30 min at 37 C followed by re-extraction of RNA with GITC as above. Cellular or tissue total RNA was extracted using TRIzol with an additional phenol/chloroform extraction step and concentrated by ethanol precipitation. The cDNA library preparation for plasma and serum exRNAs was done using a multiplexed (up to 24 samples) small RNA-seq cDNA library preparation protocol (Hafner et al., Methods, 2012, doi: 10.1016/j.ymeth.2012.07.030) using a 19- and 45-nt size selection relative to input RNA. Total RNA isolated from cells, platelets, or whole tissue samples was processed using the Illumina Stranded mRNAseq TruSeq kit following the manufacturers instructions but using Protoscript II reverse transcriptase (NEB, cat# ML0368L). Sequencing was conducted in the Genomics Core Facility at The Rockefeller University  on a Hiseq 2500 sequencer. The read processing and annotation for small RNA-seq of serum and plasma samples followed a hierarchical annotation according to  Farazi et al. (Methods, 2012; doi: 10.1016/j.ymeth.2012.07.020) with modifications in the hierarchy and transcriptome. Long RNAseq reads from tissues or cells were aligned to the human genome reference build 38 using the STAR aligner (2.4.2a) (13) allowing up to two mismatches. Count matrices were generated using the featureCounts program(14) from the Subread software suite (v1.5.1) based on Ensembl release 88, summarized on a gene level, and fractionally counting multi-mapping reads but ignoring reads overlapping more than one annotation feature.