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| Status |
Public on Apr 13, 2018 |
| Title |
Plasma 2 mL Illumina TruSeq |
| Sample type |
SRA |
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| Source name |
Plasma
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| Organism |
Homo sapiens |
| Characteristics |
cell type: Plasma
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| Growth protocol |
A large pool of plasma (30 mL) was used for comparison of kits and input volumes. Whole blood was collected from normal healthy volunteers in EDTA tubes, spun at 2500 rpm to separate plasma. Plasma was pooled, and 1 mL aliquots placed in tubes and stored at –80° C. The appropriate number of tubes were combined and isolated for each comparison. For example, for the 5 mL input volume, 5 aliquots were selected and the RNA isolated (using miRVana Paris, ThermoFisher AM1556, with modifications [25] and sample preparation for sequencing performed.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated (using miRVana Paris, ThermoFisher AM1556.
Illumina TruSeq Small RNA Library Preparation: Small RNA libraries were generated using Illumina TruSeq Small RNA Sample kit (RS-200-0048; Illumina). The reagents were utilized with the following modifications. The 3’ adapter, 5’ adapter, Stop Solution, RNase Inhibitor and RT primer were diluted by 50% with water [25]. During PCR amplification, the Index primer and RNA PCR primer volumes were reduced by 50% and the volume replaced with water. PCR amplification was performed based on the upper and lower recommendation of the kits. A total of 16 cycles for the 10 ng starting material and 11 cycles for the 1 μg starting material. The libraries were then run in a 6% TBE Gel for 55 minutes at 140 volts and the between 140 to 160bp were excised from the gels. These gel pieces were fractured into smaller pieces and allowed to incubation on a rotator overnight in water. Then an ethanol precipitation was performed to precipitate the RNA and the resulting pellet of RNA was resuspended in 11ul of ultra pure water. BiooScientific NEXTFlex Small RNA Library Preparation: Small RNA libraries were generated using NEXTFlex Small RNA Library Prep Kit v2 (Cat #5132-03). The manufacturer’s instructions where followed through PCR amplification. The optional stop point was used post the RT reaction prior to clean up and PCR amplification. In regards to PCR amplification the 1 μg samples received 12 cycles and the 10 ng sample received 18 cycles. Following PCR amplification, the libraries where then run in a 6% TBE Gel for 30 minutes at 200 volts and the between 150 to 170bp were excised from the gels. At this point there was a divergence from the manufactures instruction, an ethanol precipitation was used in place of the recommenced bead purification. The resulting pellet of RNA was resuspended in 11 μl of ultra pure water.
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| Library strategy |
ncRNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
Total extracellular RNA
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| Data processing |
The raw sequence image files from the Illumina HiSeq 2500 or Illumina MiSeq in the form of .bcl are converted to the fastq format using bcltofastq v1.8.4 and checked for quality to ensure the quality scores do not deteriorate drastically at the read ends. The adapters from the 3’ end are clipped using cutadapt v.1.10 [28] (http://cutadapt.readthedocs.io/en/stable/guide.html). Reads shorter than 15 nts are discarded and after adapter trimming, the 3’ bases below a quality score of 30 are trimmed as well.
Reads that arise from human rRNA and contamination from library preparation protocols are removed before they are mapped to the human genome. The reads are first mapped to a library of UniVec contaminants, a collection of common vector, adapter, linker and PCR primer sequences collated by the NCBI. They are then mapped to human rRNA sequences obtained from NCBI The reads are mapped to the rRNA and UniVec sequences using Bowtie2 [29] and those that map are removed from the analysis. The alignment to the human genome and transcriptome takes place in two stages. First, the rRNA and UniVec free reads are mapped to the human genome (hg19) using STAR [30]. The reads that map to the genome are then mapped to the human transcriptome. Also, the reads that are not mapped to the human genome are mapped to the human transcriptome, The library for the human transcriptome is built by concatenating miRNAs and hairpins from miRBase 21 [31], tRNAs from gtRNAdb [32], piRNAs from piRBase v1.0 [33], protein-coding, non-coding and and other RNA sequences from ENSEMBL 75. The STAR alignment is performed end to end with a single mismatch allowed while mapping and each read is allowed to multimap to at most 40 RNA annotations. Here, there is no mismatch allowed and each read is allowed to multimap to at most 40 RNA annotations.
Genome_build: hg19
Supplementary_files_format_and_content: text files with normalized counts
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| Submission date |
Apr 12, 2018 |
| Last update date |
Apr 13, 2018 |
| Contact name |
SAUMYA Das |
| E-mail(s) |
[email protected]
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| Organization name |
MGH
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| Street address |
185 Cambridge Street
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02114 |
| Country |
USA |
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| Platform ID |
GPL24878 |
| Series (1) |
| GSE113044 |
Plasma small RNASeq comparison with Illumina TruSeq and Biooscientific NextFlex |
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| Relations |
| BioSample |
SAMN08918751 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3094429_2ml_Truseq_norm_counts.txt.gz |
10.2 Kb |
(ftp)(http) |
TXT |
| GSM3094429_2ml_Truseq_raw_counts.txt.gz |
7.1 Kb |
(ftp)(http) |
TXT |
| Processed data provided as supplementary file |
| Raw data not provided for this record |
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