title: GSM5512255: ZW2PLASMAB165A12; Homo sapiens; miRNA-Seq
library_construction_protocol: Low speed centrifugation (2500 g x 15 minutes, room temperature) was performed, then transfer of supernatant, 2nd round of centrifugation (same parameters), then transfer of supernatant. A self-developed method and self-developed solutions were used for nucleic-acid extraction, nucleic-acid partitioning, and column purification (RNA isolation), see Max et al. (2018) PNAS for details. For exRNA carrier subfractionation, selected samples were fractionated by differential ultracentrifugation into 10,000 xg pellet, 100,000 xg pellet and corresponding supernatant fractions: 11 ml of platelet-depleted plasma was centrifuged at 10,000 × g in 12 ml tubes (Sorvall, #03699) for 30 min. The supernatant was transferred by aspiration into a fresh 15 ml tube and mixed by inversion. Two aliquots of 500 µl of this supernatant were transferred into 1.5 ml microcentrifuge tubes for later RNA isolation. The 10,000 × g fragile pellet was suspended in 2.0 ml of phosphate-buffered saline (PBS), transferred into a 2.0 ml microcentrifuge tube, and centrifuged at 10,000 × g in a microcentrifuge for 30 min. The PBS supernatant was removed by aspiration, and the residual ≤10 µl volume pellet was resuspended in 450 µl PBS. The residual 10 ml of 10,000 × g supernatant was transferred into a 12 ml tube (Sorvall, #03699) and centrifuged at 100,000 × g for 90 min. The supernatant was transferred as described above, and two 500 µl aliquots of the 100,000 × g supernatant were transferred into 1.5 ml microcentrifuge tubes. The 100,000 × g pellet was washed with 10 ml PBS and centrifuged again at 100,000 × g for 90 min. The PBS supernatant was carefully removed as described above. The pellet was resuspended in 450 µl PBS. All pellet and supernatant fractions were flash-frozen in liquid nitrogen. Automated RNA isolation was conducted as previously described {Max:2018km} with the following modifications: Samples were organized in batches of 24 samples of 450 µl full plasma or plasma subfractions. Vesicle fractions were adjusted with PBS to 450 µl. Calibrator Set1, composed of ten 21-nt 5'-phosphorylated RNA oligos was added at an absolute amount of 2 attomoles per oligonucleotide along with the denaturant to each sample. Large cohort sample library preparations used 3' adapters, and PCR amplification methodologie, however, P12 family and plasma subfraction samples used novel 3' adapters and PCR primers to accommodate changes in Illumina flow cell design after HiSeq was discontinued. RNA equivalent to an input 225 µl plasma was spiked with calibrator Set2 composed of another ten 21-nt 5'-phosphorylated RNA oligos at an absolute amount of 1 attomol per oligonucleotide and entered the first step of the cDNA library preparation. In sample libraries of plasma subfraction RNA from 225 µl supernatant or 5 ml pellet was used to which 0.2 attomoles per oligonucleotide of calibrator Set2 was added. After the 3' ligation, up to 24 samples were multiplexed, size-selected by denaturing 15%, ligated to 5' adapter, reverse-transcribed and PCR-amplified. To enable comparisons with earlier results (28), the PAGE size-selection limits were maintained at 19 to 45 for the large (n = 496) and P12 family cohorts (n = 12 samples). After discovering that miRNAs were noticeably underrepresented in the 10K and 100K pellet fractions, the size range for all plasma subfraction libraries (4 plasma subfractions, n = 17 samples, each) was adjusted to 19 to 24 nt in favor of miRNAs. cDNA libraries were sequenced as single-lane high-output single-read 75-bp HiSeq 2000 (Large cohort, n = 496 samples) or as Nextseq-500 100-bp single-read runs (P12 family, n = 12 samples; 4 Plasma subfractions, n = 17 samples each). Sequencing data were processed using an in-house RNA-sequencing data analysis platform (RSDAP) using extraction lenth of 16 – 45 nt, a minimum 3' overlap of adapter with 0/1 mismatches of 4/5 nt, respectively, removal of low complexity sequences. For read annotations, profile annoDB_jul2014c was used, which included reference sequences for definitions for human and virus RNA sequences based on miRBASE 18 with modifications. Calibrator sequences (labeled by set) are: >Set1_cali_01_rc TCCACGACGTCTCATGTATTTC, >Set1_cali_04_rc GGGTACCATACCGGTTGTCTTA, >Set1_cali_17_rc TCATGAGTCCGTACCTTGATTG, >Set1_cali_18_rc ATCATTTACGATTCGGAGCTGT, >Set1_cali_20_rc GATAGTTCGGGATCGCTGTAAC, >Set1_cali_24_rc TGCTACTCCGATCTTTAGCCTC, >Set1_cali_25_rc AGGGCCCTTTAGGCACTAATAG, >Set1_cali_27_rc GTAGCTGTCAGTACGTTCGTGC, >Set1_cali_43_rc TCTAGTTGCGTGATGGAGAGAA, >Set1_cali_44_rc AGCCGCATTTCGTAGTGATATT, >Set2_cali_07_rc GTCCCACTCCGTAGATCTGTTC, >Set2_cali_11_rc GATGTAACGAGTTGGAATGCAA, >Set2_cali_12_rc TAGCATATCGAGCCTGAGAACA, >Set2_cali_14_rc CATCGGTCGAACTTATGTGAAA, >Set2_cali_15_rc GAAGCACATTCGCACATCATAT, >Set2_cali_16_rc TCTTAACCCGGACCAGAAACTA, >Set2_cali_26_rc AGGTTCCGGATAAGTAAGAGCC, >Set2_cali_28_rc TAACTCCTTAAGCGAATCTCGC, >Set2_cali_31_rc AAAGTAGCATCCGAAATACGGA, >Set2_cali_35_rc TGATACGGATGTTATACGCAGC