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Sample GSM4005317

Query DataSets for GSM4005317

Status

Public on Jan 15, 2020

Title

03dFFAC0

Sample type

SRA

Source name

articular cartilage

Organism

Equus caballus

Characteristics

age: 3 days
Sex: female
tissue: articular cartilage
location: metatarsophalangeal joint
individual: foal_2

Extracted molecule

total RNA

Extraction protocol

Samples were collected immediately following euthanasia and were flash frozen in liquid nitrogen or placed in RNAlater (Qiagen, Valencia, CA) at 4°C for 24-48 hours prior to storage at -80°C. Frozen samples were pulverized and placed in TRIzol reagent (Invitrogen, Carlsbad, CA) prior to mechanical homogenization. RNA extraction was performed on spin columns using the RNeasy Micro Kit (Qiagen, Valencia, CA) per manufacturer instructions.
Standard library preparation and sequencing (100 base-pair, paired-end) on an Illumina Hi-Seq sequencer (90 samples on a Hi-Seq 2500, 22 samples on a Hi-Seq 4000)

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Data processing

Quality control on all 56 samples (112 paired-end files) was performed using fastp (version 0.19.5) and summarized using MultiQC (version 1.6)
All fastq files were trimmed using Trimmomatic(version 0.38) in paired end mode to trim any remaining adaptors, then trim leading or trailing bases with quality scores below 28, then discarding a read pair if either read was shorter than 30 bases
Salmon (version 0.11.3) was used to quasi-map reads in paired-end mode to quantify transcript abundance (with additional arguments –seqBias --gcBias –numBootstraps=30) to the EquCab3.0 genome assembly utilizing NCBI Annotation Release 103
R package tximport was used to estimate gene-level counts from transcript-level quantification. For the articular cartilage tissue (AC0), two individual horses had two samples taken at slightly different locations (ACD vs. ACS) in the stifle joint. Gene-level counts for the ACD and ACS samples from the same horse were added together at the gene-level as they looked extremely similar to each other.
Reads were filtered and normalized (TMM) using edgeR, surrogate variables were calculated and counts were corrected using sva, and DE analysis performed using limma-trend
Genome_build: EquCab3.0
Supplementary_files_format_and_content: tab-delimited files with normalized log2(cpm) values for 54 of 56 samples

Submission date

Aug 02, 2019

Last update date

Jan 15, 2020

Contact name

Ann Marie Kemper

E-mail(s)

amkemper@illinois.edu

Organization name

University of Illinois

Street address

1008 W Hazelwood Dr