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Sample GSM2690584

Query DataSets for GSM2690584

Status

Public on Oct 18, 2017

Title

EX4-Treated-2

Sample type

SRA

Source name

transplanted human juvenile islet grafts

Organisms

Homo sapiens; Mus musculus

Characteristics

strain: Recipient - NSG mice
tissue: Human islet grafts with some mouse kidney tissue
age: Donor - 6 years old
genotype: recipient mice - immunodeficiency
treatment: Ex4

Treatment protocol

Two weeks after human islet transplantation, Ex-4 or PBS was delivered by micro-osmotic pumps implanted in recepient mice for 4 weeks.

Extracted molecule

total RNA

Extraction protocol

Norgen Total RNA Purification Kit
Five hundred nanograms of total RNA was required for downstream RNA-seq applications. Polyadenylated RNAs were isolated using NEBNext Magnetic Oligo d(T)25 Beads. The NEBNext mRNA Library Prep Reagent Set for Illumina (New England BioLabs Inc., Ipswich, MA, USA) was then used to prepare individually bar-coded next-generation sequencing expression libraries as per manufacturer's recommended protocol. Library quality was assessed using the Qubit 2.0 Fluorometer, and the library concentration was estimated by utilizing a DNA 1000 Chip on an Agilent 2100 Bioanalyzer. Accurate quantification for sequencing applications was determined using the qPCR-based KAPA Biosystems Library Quantification Kit (Kapa Biosystems, Inc., Woburn, MA, USA). Paired-end sequencing (100 million, 100-bp, paired-end reads) was performed on an Illumina HiSeq2500 sequencer (Illumina, Inc., San Diego, CA, USA).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

Human islet grafts + mouse kidney tissue contaminated

Data processing

Post-processing of the sequencing reads from RNA-seq experiments for each sample was performed using HudsonAlpha’s unique in-house RNA-seq data analysis pipeline. Briefly, quality control checks on raw sequence data for each sample were performed using FastQC (Babraham Bioinformatics, Cambridge, UK). Raw reads were mapped to the reference hg19 using TopHat v2.0. The alignment metrics of the mapped reads were estimated using SAMtools. Aligned reads were imported to the commercial data analysis platform AvadisNGS (Strand Scientifics, CA, USA). After quality inspection, the aligned reads were filtered on the basis of read quality metrics; reads with a base quality score of less than 30, alignment score of less than 95, and mapping quality of less than 40 were removed. Remaining reads were then filtered on the basis of their read statistics; missing mates, translocated, unaligned, and flipped reads were removed. The reads list was then filtered to remove duplicates. Samples were grouped and transcript abundance was quantified for this final read list using Trimmed Means of M-values (TMM) as the normalization method. Output data utilized for all subsequent comparisons were summarized as normalized signal values generated by AvadisNGS. Differential expression of genes was calculated on the basis of fold changes (using the default cut-off ≥ ±2.0) observed in comparisons between defined conditions, and the p-value of the differentially expressed gene list was estimated by ANOVA using Benjamini Hochberg corrections of 0.05 for false-discovery rate.
Genome_build: hg19
Supplementary_files_format_and_content: Excel files include RPKM values for each Sample

Submission date

Jun 29, 2017

Last update date

May 15, 2019

Contact name

NRIPESH PRASAD

E-mail(s)

nprasad@hudsonalpha.org

Phone

2563279413

Organization name

HUDSONALPHA INSTITUTE FOR BIOTECHNOLOGY