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Sample GSM1726440

Query DataSets for GSM1726440

Status

Public on Jan 05, 2016

Title

D1-2_tot_100ss

Sample type

SRA

Source name

human primary granulocytes

Organism

Homo sapiens

Characteristics

cell type: granulocyte
tissue: blood
passages: primary

Extracted molecule

total RNA

Extraction protocol

Primary Granulocytes were isolated from fresh blood collected in the morning using Ficoll density centrifugation method. After erythrocyte depletion and washing in ice cold PBS (supplemented by 2mM EDTA) granulocytes were pelleted and immediately lyzed using TRI-reagent (Sigma-Aldrich T9424) and RNA was then isolated using the manufacturers protocol. Total RNA was DNaseI treated using the DNA-FreeTM kit (Ambion).
RNA for total RNA-seq was depleted for Ribosomal RNA using the RiboZero rRNA removal kit (Human/Mouse/Rat) (Epicentre) according to manufacturer's protocol. RNA for PolyA+ RNA-seq was enriched for polyadenylated RNA using TruSeq RNA Sample Prep Kit v2 (Illumina). Pretreeated RNA was then subject to library preparation. Strand-specific RNA-seq libraries were prepared employing the TruSeq RNA Sample Prep Kit v2 (Illumina) modified to obtain strand-specificity using dUTP incorporation and UDGase treatment. RNA-seq was performed by the Biomedical Sequencing Facility (BSF) in Vienna using the Illumina HiSeq 2000 platform.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

granulocyte_denovo_lncRNA_loci_RPKM_total_gra_7donorsx3replicates.xls, granulocyte_denovo_lncRNA_loci_variation_analysis.xls, granulocyte_denovo_lncRNA_RPKM_total_gra_7donorsx3replicates.xls, granulocyte_denovo_lncRNAs_variation_analysis.xls, granulocyte_denovo_mRNA_loci_RPKM_total_gra_7donorsx3replicates.xls, granulocyte_denovo_mRNA_loci_variation_analysis.xls, granulocyte_denovo_mRNA_RPKM_total_gra_7donorsx3replicates.xls, granulocyte_denovo_mRNAs_variation_analysis.xls,

Data processing

RNA sequencing data was aligned to the hg19 human genome using STAR (version 2.3) (See Kornienko, et al , Methods for details)
de novo lncRNA and mRNA annotations were obtained using Cufflinks, Cuffmerge and a series of filtering steps (described in Kornienko et al) for granulocytes (using granulocytes PolyA+ RNA-seq deposited here)
RPKMs were calculated using RSeQC package
Genome_build: hg19
Supplementary_files_format_and_content: deposited are expression levels (RPKM) of granulocyte lncRNA and mRNA transcripts and loci in 21 total granulocyte samples (used for variation analyses), excel table with first 6 column indication transcript/locus coordinate, name and strand, and next columns - RPKM values. Name of the column corresponds to the name of the sample.
Supplementary_files_format_and_content: "_variation analysis.xls" excel tables contain the position, name and the strand of the transcript / locus and data used to plot
Supplementary_files_format_and_content: "_novelty_and_position_class" are tables with the names of granulocyte de novo lncRNA loci/transcripts and their classification according to novelty (as shown in Fig 3A, Kornienko et al), or position relative to protein coding genes (as shown in Fig1C).
Supplementary_files_format_and_content: "_annotation" files are in BED12 format and contain de novo lncRNA and mRNA annotations obtained in the study for granulocytes and LCL

Submission date

Jun 29, 2015

Last update date

May 15, 2019

Contact name

Aleksandra E Kornienko

E-mail(s)

[email protected]

Organization name

CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences

Lab

Barlow lab