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Sample GSM1438894

Query DataSets for GSM1438894

Status

Public on Dec 01, 2015

Title

Sample type

SRA

Source name

Neutrophil

Organism

Homo sapiens

Characteristics

cell type: Neutrophil isolated from peripheral blood

Treatment protocol

Growth protocol

Extracted molecule

total RNA

Extraction protocol

For each participant 20 mL of peripheral blood was collected into heparinized tubes. Enrichment for neutrophil was performed by a Dextran-Ficoll sedimentation and centrifugation method60. Briefly, 20 mL of peripheral blood was mixed with Dextran-RPMI media in a 4:1 ratio. After 40 minutes incubation at RT, the upper layer (white blood cell-rich plasma) was layered onto Ficoll (aspirate: Ficoll of 2:1 ratio) and centrifuged for 15 min at 2500 rpm. To remove the remaining erythrocytes, the cell pellet was treated with 2 mL ddH2O for 10-15 seconds, after which 25 mL of RPMI was added. The suspension was centrifuged for 5 min at 1400 rpm and the cell pellet was resuspended in 20 mL phenol red-free RPMI-1640. An aliquot of this suspension was used to check presence of dead cells with trypan blue staining. This preparation contained > 98% neutrophils.
Total RNA was isolated with RNeasy Mini Kit (Qiagen) following manufacturer’s protocol. Two rounds of RNase-free DNase I digestion (Qiagen) was performed to eliminate genomic DNA during the extraction process. RNA concentrations were determined using NanoDrop 2000 (Thermo Scientific). The quality and integrity of the RNA was determined using the RNA 6000 Pico chip on 2100 Bioanalyzer (Agilent Technologies). The median RNA integrity number (RIN) for the 4 samples was 8.05. RNA libraries were constructed using 1 µg of total RNA with TruSeq stranded mRNA Sample Preparation kit (Illumina, USA) following the manufacturer’s protocol.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Data processing

RNA-seq library from 4 individuals were sequenced in Illumina HiSeq Machines, Base-calling perfromed using RTA software and demultiplexed to obtain individual Fastq files.
Alignment of the sequenced reads were perfromed using Tophat package
Differential expression analysis was perfomed using cufflink and cuffdiff packages
Exon usage analysis was perfromed using DEXSeq R package.
Genome_build: GRCh37
Supplementary_files_format_and_content: Excel file contains FPKM values of 4 samples (excel)

Submission date

Jul 17, 2014

Last update date

May 15, 2019

Contact name

Aniruddha Chatterjee

E-mail(s)

aniruddha.chatterjee@otago.ac.nz

Phone

+64-210701558

Organization name

University of Otago