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Sample GSM6745978

Query DataSets for GSM6745978

Status

Public on Jan 31, 2024

Title

NSTEMI_14_reanalyzed

Sample type

SRA

Source name

Whole blood

Organism

Homo sapiens

Characteristics

tissue: Whole blood
Sex: Male
age: 58
acs: NSTEMI
avsc: No

Growth protocol

Blood samples were collected and mantained into Tempus Blood RNA tubes at -80 °C until processing, following manufacturer's instructions.

Extracted molecule

total RNA

Extraction protocol

Total RNA was isolated using the Tempus Spin RNA Isolation Kit (Applied Biosystems) and was treated with RNase-free DNase-I to eliminate genomic contamination, following the manufacturer’s instructions.
Five µg of total RNA were precipitated, α- and β-globin mRNAs were depleted with GLOBINclear Whole Blood Globin Reduction kit (Applied Biosystems), and poly(A)+ RNA was enriched following MicroPoly(A) Purist kit protocol (Applied Biosystems). Libraries were prepared and pooled together using the multiplex SOLiD System Sequencing and Barcoding kits. Complementary DNA (cDNA) amplification reaction was conducted with 20 µL template in 100 µl end-volume reaction and with 16 PCR cycles. Clonally amplification of library templates was performed on SOLiD p1 DNA beads by emulsion PCR (ePCR) using 0.5 pM library template and E120 EZbeads scale.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

AB 5500xl Genetic Analyzer

Description

GSM2756729

Data processing

We used XSQTools (Applied Biosystems) with default parameters to remove reads with low quality base values and to generate .csfasta and .QV.qual files for each samples. Fiannly, cs.fasta and .QV.qual files were converted into .fastq format.
Sequential aligning of raw reads was performed against the GRCh38 Human Genome reference (release 99) with the “Spliced Transcripts Alignment to a Reference” (STAR v. 2.7.5c) software, and with “Bowtie2” (v. 2.4.1) to align locally any reads not mapped by STAR. We excluded 'haplotypes' and 'patches' sequences from the reference, in order to focus on primary assembly and to avoid under-estimation of gene expression.
We implemented the reference annotation based transcript (RABT) procedure, using StringTie suite v2.1.4 to create a new assembly for downstream analysis, integrating the information about known genes and transcripts position in the genome (Ensemble GTF release 99) with those reads mapped in intergenic or intronic regions.
Gene expression quantification were computed by “featureCounts” (v.2.0.1) grouping meta-features by 'gene name'.
Assembly: HG38/GRCh38.99
Supplementary files format and content: Tab-delimited text files include raw counts values for each Sample. The header of the files reports gene name, Ensemble gene ID, chromosome location, gene length, biotype, biotype class and sample identifiers.

Submission date

Nov 21, 2022

Last update date

Jan 31, 2024

Contact name

Gualtiero Ivanoe Colombo

E-mail(s)

gualtiero.colombo@cardiologicomonzino.it

Phone

+39 0258002464

Organization name

Centro Cardiologico Monzino IRCCS