Project description:Purpose: To identify differential mRNA expression in response to SHH stimulation in murine embryonic and lung fibroblasts Methods: NIH-3T3 and MLg cell lines were treated with recombinant SHH or vehicle control and RNA-seq was performed using Illumina Miseq Nano (performed by Admera Health, LLC (South Plainfield, NJ)). The FASTQ files were checked for quality using FastQC (v0.11.2). FASTQ files were mapped to the mm10 assembly of the mouse genome using TopHat; fragments per kilo base per million mapped reads (FPKM) calculation and differential expression analysis were performed using Cufflinks (v 2.2.1). Results: Comparison of differentially up-regulated and down-regulated mRNAs of secretory protein genes in NIH-3T3 and MLg cell lines upon SHH activation revealed interesting differences in the production of some ligands.
Project description:E15.5 embryos were micro-disscted from Gata4 G295S mutant mice and littermate controls, RNA was isolated using Norgen total RNA isolation, and libraries were generated with the RNA TruSeq Stranded Total RNA kit. 50 base pair paired end reads were obtained on an illumina high seq 2500. Fastq files were aligned to the mouse genome using STAR aligner. QC was performed using RNASeQC and RSeQC. BAM files were processed using cufflinks pipeline.
Project description:Transcriptomic profile of human adipose tissue progenitor cells was performed as follows. For AmpliSeq transcriptome sequencing library construction, AmpliSeq™ Library PLUS, AmpliSeq Transcriptome Human Gene Expression Panel and AmpliSeq CD indexes SetA kits were purchased from Illumina and sequencing libraries were constructed as described in AmpliSeq for Illumina Transcriptome Human Gene Expression Panel reference guide (Illumina). Equimolar concentrations of libraries were pooled at 4 nM and denatured and diluted as described in Denature and Dilute Libraries Guide (Illumina) and adjusted to final concentration of 1.4 pM. Resulting library was sequenced on NextSeq 500 using NextSeq 500/550 High Output v2 kit with 2 X 151 bp cycle. Generated raw files were converted to FASTQ files and used for data analysis. AmpliSeq transcriptome FASTQ files were analyzed on Array studio V10.0 (Omicsoft, Qiagen). Following raw read QC, first and last 10 bases were trimmed and mapped to reference genome Human.B38. The read count data was generated using GeneModel RefGene20170606. Resulting data was normalized by DESeq package, transformed to log2 value and used for ANOVA analyses.