Computational protocol: Transcriptome Sequencing Reveals Differences between Primary and Secondary Hair Follicle-derived Dermal Papilla Cells of the Cashmere Goat (Capra hircus)

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Protocol publication

[…] Cells were seeded on 24-well culture plates at 1.0×104 cells/ml. Cell numbers and cell density of each well were counted and recorded daily. The control comprised dermal fibroblast cells (DFCs), which were also obtained from Inner Mongolia Cashmere goat during the anagen phase. Cell numbers in four wells were counted at each time point, and the averages were used to plot the cell growth curve. Systat SigmaPlot 12.3 (http://www.systat.com/) plotted the curve. The mean population doubling times were estimated for the period of most rapid growth (between 3 and 19 days for DPCs and between 3 and 6 days for DFCs) and calculated as described by Oliver et al. Each experiment was repeated three times. [...] About 1.0×107 PHF-DPCs and 1.0×107 SHF-DPCs at the second passage were obtained from the six groups of cultured PHF-DPCs and six groups of cultured SHF-DPCs, respectively. For each sample, cells were collected averagely from the different groups and pooled together. Total RNAs were isolated from each sample using a TRIzol Plus RNA Purification Kit according to the manufacturer's protocol (Invitrogen, Carlsbad, California, USA). Total RNA purity and concentration were determined using a 2100 Bioanalyzer Nanochip (Agilent Technologies, Palo Alto, CA, USA). The RNA-Seq libraries were constructed as previously described . An Illumina/Solexa HiSeq2000 platform was used to sequence the RNA-Seq libraries. The raw reads were filtered to remove the adaptor sequences, low quality reads (>2% base smaller than Q20 per read) and reads containing undetermined bases (>2% 'N's per read was removed). The cleaned, high quality reads from PHF-DPCs and SHF-DPCs were aligned against the Capra hircus genome (NCBI PRJNA158393) assembly using TopHat . Cufflinks was used to generate transcript annotation files and Cuffdiff , , was used to measure the fragments per kilobase of transcript per million fragments mapped (FPKM) value for each protein-coding gene in the two types of DPCs. The differentially expressed genes between two samples were selected using the following criteria: i) if the FPKM value for a certain gene in both samples was greater than 1, the difference between them should be at least twofold. ii) If the FPKM value for a certain gene in one sample was less than 1, the FPKM value for this gene in the paired sample should be greater than 2. The goat genome assembly, genome annotation file and protein-encoding gene sequence can be obtained from the Goat Gene Database (http://goat.kiz.ac.cn/GGD/). The initial Illumia short reads generated by HiSeq2000 system in this study have been submitted to the NCBI Sequence Read Archive (SRA) under accession numbers SRX327891 (PHF-DPCs) and SRX327892 (SHF-DPCs). […]

Pipeline specifications

Software tools SigmaPlot, TopHat, Cufflinks
Applications Miscellaneous, RNA-seq analysis
Organisms Capra hircus
Diseases Papilledema