Extracellular Vesicle (EV) RNA-Sequencing

“Salmon is a method for quantifying transcript abundance from RNA-seq reads that is RNA Strandaccurate and fast. Salmon uses new algorithms to provide accurate expression estimates quickly while using little memory. Salmon performs its inference using an expressive and realistic model of RNA-seq data that takes into account experimental attributes and biases commonly observed in real RNA-seq data.

Salmon v0.11.3

      Options: quant

      --libTypeA

      --threads 16 --numBootstraps 100

      --seqBias --gcBias

      --dumpEq --geneMap

      --gencode.v29.primary_assembly.annotation.gtf

STAR (Spliced Transcripts Alignment to a Reference) aligns high-throughput long and short RNA-seq data to a reference genome using uncompressed suffix arrays. STAR is a stand alone software capable of aligning reads in a continuous streaming mode. It is able to detect canonical junctions, non-canonical splices and chimeric transcripts and to map full-length RNA sequences.

STAR v2.6.1d

       STAR --genomeDir STARREF --runMode alignReads

      --twopassMode Basic\

      --outFileNamePrefix SAMPLEID --readFilesCommand zcat\

      --readFilesIn FASTQL1 FASTQL2

      --outSAMtype BAM SortedByCoordinate\

      --outFilterType BySJout --outFilterMultimapNmax 20\

      --outFilterMismatchNmax 999

      --outFilterMismatchNoverLmax 0.1\

      --alignIntronMax 1000000 --alignMatesGapMax 1000000\

      --alignSJoverhangMin 8 --alignSJDBoverhangMin 1\

      --chimOutType WithinBAM --chimSegmentMin 15\

      --chimJunctionOverhangMin 15 --runThreadN 16\

      --outSAMstrandField intronMotif

      --outSAMunmapped Within\

      --outSAMattrRGline RGTAGLIST

featureCounts is a read summarization program suitable for counting reads generated from either RNA or genomic DNA sequencing experiments. The program is developed for counting reads to genomic features such as genes, exons, promoters and genomic bins.”

Feature Counts v1.6.2

      Options:

      --T 2 -p  -t exon  -g gene_id  

      --a gencode.v19.annotation.patched_contigs.gtf

      --s 2

Kits that isolated all extracellular RNA were utilized for this experiment – total extracellular RNA preparation. Samples were then processed in the same way as the existing AMP PD whole blood transcriptomics samples.

CSF RNA Isolation
For each CSF subject sample, 1mL of CSF was thawed on ice. RNA was then isolated with Qiagen’s miRNeasy Serum/Plasma Kit (Qiagen, Cat. No. 217184) using a modified protocol to include an on-column DNase treatment (Qiagen, Cat. No. 79256).
 
For each CSF pool, 5mL of CSF provided in 200uL aliquots was thawed on ice, then pooled together, gently mixed, and re-aliquoted into five 1mL aliquots. RNA was then isolated from each 1mL CSF aliquot with Qiagen’s miRNeasy Serum/Plasma Kit (Qiagen, Cat. No. 217184) using a modified protocol to include an on-column DNase treatment (Qiagen, Cat. No. 79256). Isolated RNA across all five aliquots was then pooled together, gently mixed, and re-aliquoted evenly across 5 microcentrifuge tubes. 

Plasma RNA Isolation
For each of 18 plasma subject samples, 1mL of plasma was thawed at room temperature, then immediately moved to ice. RNA was then isolated with Qiagen’s miRNeasy Serum/Plasma Kit (Qiagen, Cat. No. 217184) using a modified protocol to include an on-column DNase treatment (Qiagen, Cat. No. 79256).
 
For each of 169 plasma subject samples, 1mL of plasma was thawed at room temperature, then immediately moved to ice. RNA was then isolated with Norgen Biotek’s Plasma/Serum Circulating and Exosomal RNA Purification Mini Kit - Slurry Format (Norgen Biotek, Cat. No. 51000) with DNase treatment (Norgen Biotek, Cat. No. 25720).
 
For each plasma pool, 5mL of plasma provided in 200uL aliquots was thawed at room temperature then immediately moved to ice. All aliquots were pooled together, gently mixed, and re-aliquoted into five 1mL aliquots. RNA was then isolated from each 1mL plasma aliquot with Norgen Biotek’s Plasma/Serum Circulating and Exosomal RNA Purification Mini Kit - Slurry Format (Norgen Biotek, Cat. No. 51000) with DNase treatment (Norgen Biotek, Cat. No. 25720). Isolated RNA across all five aliquots was then pooled together, gently mixed, and re-aliquoted evenly across 5 microcentrifuge tubes.  

CSF RNA Whole Transcriptome Library Preparation & Sequencing
For each CSF RNA sample, a uniquely dual-indexed, Illumina-compatible, double-stranded cDNA whole transcriptome library was synthesized from all total RNA in 1mL of CSF with Takara Bio’s SMART-Seq Stranded Kit Components (Takara Bio, Cat. No. 634447) and SMARTer RNA Unique Dual Index Kit (Takara Bio, Cat. No. 634452 & 634457). A replicate of each CSF pool was prepared in parallel with every batch of subject CSF. Briefly, this library preparation included RNA priming (72 °C for 3 min), a 5-cycle Indexing PCR, ribosomal cDNA depletion, and a 16-cycle enrichment PCR. 

Each library was measured for size with Agilent’s High Sensitivity D1000 ScreenTape and buffer (Agilent, Cat. No. 5067-5584 & 5067-5603) and concentration with KAPA SYBR FAST Universal qPCR Kit (Roche, Cat. No. KK4824). Two libraries did not produce quantifiable libraries and were not sequenced. Libraries were then combined into an equimolar pool which was also measured for size and concentration. The pool was then normalized to 2 nM with a 1% v/v PhiX Control v3 spike-in (Illumina, Cat. No. FC-110-3001), denatured and further diluted, loaded into a NovaSeq 6000 flow cell cartridge (Illumina, Cat. No. 20028313), and sequenced at 101 x 9 x 9 x 101 cycles with standard workflow and a final flow cell concentration of 400 pM. Libraries were sequenced to at least 50 M read pairs (or 100 M paired-end reads).

Plasma RNA Whole Transcriptome Library Preparation & Sequencing
For each plasma RNA sample, a uniquely dual-indexed, Illumina-compatible, double-stranded cDNA whole transcriptome library was synthesized from up to 2ng of total plasma RNA with Takara Bio’s SMART-Seq Stranded Kit Components (Takara Bio, Cat. No. 634447) and SMARTer RNA Unique Dual Index Kit (Takara Bio, Cat. No. 634452 & 634457). A replicate of each plasma pool was prepared in parallel with subject samples. Briefly, this library preparation included RNA fragmentation (85 °C for 2 min), a 5-cycle Indexing PCR, ribosomal cDNA depletion, and a 16-cycle enrichment PCR. Each library was measured for size with Agilent’s High Sensitivity D1000 ScreenTape and buffer (Agilent, Cat. No. 5067-5584 & 5067-5603). 1uL of each library was combined into a non-equimolar pool which was then measured for size via TapeStation and concentration via Roche’s KAPA SYBR FAST Universal qPCR Kit (Roche, Cat. No. KK4824), diluted to 70 pM, then loaded into an iSeq flowcell cartridge (Illumina, Cat. No. 20031371) with a 1% v/v PhiX Control v3 spike-in (Illumina, Cat. No. FC-110-3001), and sequenced at 101 x 8 x 8 x 101 cycles. Passing filter cluster counts per library were generated from this data and used to make a re-balanced pool which was subsequently measured for size and concentration, diluted to 2 nM with a 1% v/v PhiX Control v3 spike-in, denatured and further diluted, loaded into a NovaSeq 6000 flow cell cartridge (Illumina, Cat. No. 20028313), and sequenced at 101 x 9 x 9 x 101 cycles with standard workflow and a final flow cell concentration of 400 pM.Libraries were sequenced to at least 50M read pairs (or 100M paired-end reads). 

For each of the plasma subject samples, 1mL of plasma was thawed at room temperature, then immediately moved to ice. RNA was then isolated with Norgen Biotek’s Plasma/Serum Circulating and Exosomal RNA Purification Mini Kit - Slurry Format (Norgen Biotek, Cat. No. 51000) with DNase treatment (Norgen Biotek, Cat. No. 25720).

Plasma RNA samples were prepared for library generation with QIAGEN’s RNeasy MinElute Clean-up kit (74204), as follows: 5 ng of total RNA were treated with buffer RLT, and 100% ethanol. The sample was passed through a MinElute column, washed, dried, and RNA was eluted with ultra-pure water. Immediately after the clean-up, RNA was concentrated using a speed-vacuum centrifuge, and used for library preparation with Perkin Elmer’s NEXTFLEX Small RNA-Seq v3 and UDI barcodes (NOVA-5132-06). RNA was denatured at 70°C, and underwent 3’ adapter ligation for 2 hours at 25°C. NEXTFLEX Cleanup Beads were then used to remove excess free adapter, and the 5’ adapter was ligated for 1 hour at 20°C. cDNA was generated from the 3’ and 5’ ligated RNA, followed by a second bead clean-up, and finally PCR-amplified using UDI primers, for 18 cycles.
Libraries were size-selected and cleaned up using PAGE and the DNA Clean and Concentrate kit (Zymo, D4014). Briefly, samples were separated onto 6% PA gels, the band of interest was excised, and the gel piece was crushed and incubated in water overnight, with constant agitation. DNA binding buffer was added to precipitate the DNA, which was then applied to a column, washed, and eluted in ultra-pure water. Library size and concentration was determined via Agilent 2100 Bioanalyzer, using the High Sensitivity DNA kit.

Library pools were denatured with NaOH, and clustered onto flow cells at 14 pM, with 5% PhiX spike-in, using cBot instruments. Sequencing was carried out on Illumina’s HiSeq 2500 using TruSeq v3 reagents.