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Sample GSM2742014

Query DataSets for GSM2742014

Status

Public on Nov 03, 2017

Title

AD_2282del4_194

Sample type

SRA

Source name

skin

Organism

Homo sapiens

Characteristics

disease: atopic dermatitis (AD)
mutation: FLG mutation

Treatment protocol

mutation or wild-type

Growth protocol

samples from the skin

Extracted molecule

total RNA

Extraction protocol

After local anesthesia and under standard aseptic conditions, 4 mm punch biopsies were taken. After subcutaneous fat tissue removal, the skin biopsies were directly snap frozen in liquid nitrogen. To further minimize potential RNA degradation by skin RNAses, biopsies were not stored at -80° C, but directly processed for RNA extraction. Skin biopsies were homogenized in TRIZOL (Ambion, Austin, TX) and thereafter centrifuged to eliminate contaminating fat prior extraction with chloroform. After precipitation using isopropanol at -20°C, RNA was washed twice with ethanol and then re-suspended in Tris-EDTA buffer (Ambion, Austin, TX). Then, samples were incubated at 57°C for 10 min and RNA concentration was determined by using a NanoDrop (Thermoscientific, Waltham, MA). RNA quality was assessed by using a Bioanalyzer chip assay (Agilent technologies, Santa Clara, CA). Samples were stored at -80°C until RNA sequencing.
50 ng of total RNA were amplified by applying Ovation RNA-Seq System V2 (NuGen, Emeryville, CA). The resulting cDNAs were pooled in equal amount and used to prepare the DNA fragment library with SOLiD System chemistry (Life Technologies Corp., Carlsbad, CA). Sequencing was performed using SOLiD 5500W platform and DNA sequencing chemistry (Life Technologies Corp., Carlsbad, CA). Raw reads (75 bp) were color-space mapped to the human genome hg19 reference using Maxmapper algorithm implemented in the Lifescope software (Life Technologies Corp., Carlsbad, CA). Mapping to multiple locations was permitted. Reads with score less than 10 were excluded. Average mapping quality was 30. Analysis of the RNA content and gene-based annotation was done with whole-transcriptome work- flow of Lifescope.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Ion Torrent S5

Description

Ion Torrent S5

Data processing

Color-space base calling
Mapping, alignment with Lifescope
Lifescope transcriptome workflow
Genome_build: hg19
Supplementary_files_format_and_content: counts data

Submission date

Aug 14, 2017

Last update date

May 15, 2019

Contact name

Sulev Koks

E-mail(s)

sulev.koks@murdoch.edu.au

Phone

+61864570313

Organization name

Murdoch University