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Sample GSM5280911

Query DataSets for GSM5280911

Status

Public on May 04, 2021

Title

RNA_049v09001880

Sample type

SRA

Source name

Plasma

Organism

human blood metagenome

Characteristics

tissue: Plasma
cohort_number: 1
batches_per_cohort_per_source: No
geographical_location: USA: Cleveland, Ohio
cd4 count: 276
cd8 count: 609.5
cd4: cd8 t cell ratio: 0.452830188679245
hiv_infected?: Yes
on_art?: Yes
subject_classification: Non_responder (>3yrs on ART)

Extracted molecule

total RNA

Extraction protocol

sorted cells: RNA was extracted from sorted cells lysed in RNAzol RT (MRC Inc., OH, USA), according to the manufacturer’s instructions. Briefly, 0.4 volume of sterile water was added to each lysate to allow aqueous and organic phase separation. Total RNA was then extracted from the aqueous phase by isopropanol precipitation. RNA pellets were washed with 70% ethanol and resuspended in sterile water. Extracted RNA was stored at -80°C until preparation of mRNA libraries. RNA yield and integrity were verified by microelectrophoresis using the High Sensitivity RNA ScreenTape Kit (Agilent) on the 2200 TapeStation system (Agilent).
plasma: Circulating total RNA and DNA were isolated from frozen plasma using RNAzolBD (MRC) according to the manufacturer’s recommendations.
sorted cells: mRNA libraries were constructed as described previously (Sandler et al., 2014) using the NEBNext Ultra RNA library preparation kit. Polyadenylated transcripts were purified with oligo-dT magnetic beads, fragmented, reverse transcribed using random hexamers and incorporated into barcoded cDNA libraries based on the Illumina TruSeq platform. Libraries were validated by microelectrophoresis on a 2100 Bioanalyzer system (Agilent), quantified with Kapa Library Quantification Kits (Roche), pooled and clustered on Illumina TruSeq v2 flow cells. Clustered flow cells were sequenced in 2x75 base paired-end runs on Illumina HiSeq 2000 and HiSeq 4000.
plasma: RNA fragmentation and reverse transcription using random hexamers, the obtained transcripts, as well as the isolated plasma DNA, were individually incorporated into barcoded cDNA libraries on the basis of the Illumina TruSeq platform. Libraries were validated by microelectrophoresis on a 2100 Bioanalyzer system (Agilent), quantified using Kapa Library Quantification Kits (Roche), pooled and clustered on Illumina patterned flow cells. Clustered flow cells were sequenced on an Illumina HiSeq 4000 in 75-base paired reads. To control for contamination, four water samples were processed along with plasma samples from nucleic acids extraction through sequencing.

Library strategy

OTHER

Library source

transcriptomic

Library selection

other

Instrument model

Illumina HiSeq 4000

Description

PathSeq
Cleveland_PathSeq_TPM.xlsx

Data processing

Library strategy: PathSeq metagenome
sorted cells/plasma: Raw sequencing reads were trimmed off adapter sequence contaminants using Trimmomatic v. 0.36
sorted cells/plasma: Mapped onto the Refseq Grch38 of the human genome using the STAR RNA-Seq aligner (version 2.5.3a)
sorted cells: Gene abundance was then estimated by counting using HTSeq (version 0.9.1)
sorted cells: Transcript counts were then normalized by TMM (Trimmed Mean of M-values by correcting for the library size
plasma: Unaligned paired-end reads were then assembled into contigs using Trinity
plasma: Trinity contigs were then aligned against HG38 again to further remove host-derived sequences with BLAST+. Trinity contigs that aligned to non-reference human sequences in the NCBI nucleotide database were also removed
plasma: Remaining contigs were then aligned against the NCBI nucleotide database and quantified with Salmon
plasma: Taxonomic information was assigned for each contig and transcripts per million (TPM) values were collapsed at the species level for each sample. TPM are normalized for sequencing read depth and length for each sample, allowing for a more accurate comparisons between proportion of reads mapping to the same gene in various samples
Genome_build: Human (Refseq Grch38) and Non-human (NCBI nucleotide database and quantified with Salmon)
Supplementary_files_format_and_content: xlsx data matrix files with normalized read counts (sorted cells; sample by transcript) or TPM (plasma; sample by species abundance)

Submission date

May 04, 2021

Last update date

May 06, 2021

Contact name

Rafick-Pierre Sekaly

E-mail(s)

rafick.sekaly@emory.edu

Organization name

Emory University

Department

Pathology