Computational protocol: Tumour resistance in induced pluripotent stem cells derived from naked mole-rats

Similar protocols

Protocol publication

[…] Total RNA was extracted from NMR-fibroblasts and NMR-iPSCs using TRIzol reagent (Invitrogen) followed by Qiagen RNeasy column purification. The quality and quantity of the RNA preparations were assessed using a 2100 Bioanalyzer with an RNA 6000 Nano LabChip Kit (Agilent Technologies). Poly(A)+ RNA was selected and converted to a library of cDNA fragments (200–250 bp) with adaptors attached to both ends for sequencing using a TruSeq RNA Sample Prep Kit v2 (Illumina), as per the manufacturer's instructions. Libraries were quantified using a Bioanalyzer DNA High Sensitivity Kit (Agilent Technologies) and Kapa Library Quantification Kit (Kapa Biosystems) using an Applied Biosystems StepOne Real-Time PCR System, according to the manufacturer's instructions. The libraries were then loaded into a flow cell for cluster generation using the TruSeq Rapid SR Cluster Kit (Illumina) and sequenced using an Illumina HiSeq2500 to obtain 100-nucleotide sequences (single-end).Base-calling and Chastity filtering were performed using real-time analysis software version 1.18.61. Illumina fastq files generated using real-time analysis were trimmed with cutadapt (http://code.google.com/p/cutadapt/) to remove the Illumina Truseq adapter sequence, and sequences with ≥50 nucleotides were selected. The fastq sequences of NMR-iPS and Ms fibroblasts were separated using xenome (http://www.genomics.csse.unimelb.edu.au/product-xenome.php) from the trimmed fastq files. The NMR reference sequence files and annotation general feature format file were downloaded from BGI ftp site (ftp://ftp.genomics.org.cn/pub/Heterocephalus_glaber/).We used bowtie build v.0.12.9 to build Burrows–Wheeler transform indexes for the reference genomic sequence, which were combined with data from genomic and mitochondrial sequence files. The trimmed NMR fastq files were aligned to the reference genomic sequence using TopHat v.2.0.12 (http://tophat.cbcb.umd.edu/) with SAMtools v.0.1.18 and non-default parameters as follows: --num-threads 6 --max-multihits 1 --transcriptome-index=‘BGI naked mole-rat cdna sequence'.Transcript abundances were calculated and fragments per kilobase of transcript per million mapped reads-normalized to the upper quartile using Cufflinks v.2.0.12 (http://cufflinks.cbcb.umd.edu/). The differential expression of transcripts by fibroblasts and iPSCs was estimated using Cuffdiff. Heat maps of the differentially expressed genes were generated using the heatmap.3 function in the plots package of R. We selected 31 pluripotency-related genes and 15 fibroblast-marker genes shown in and . In , c-Myc-target genes were selected, as previously described. […]

Pipeline specifications

Software tools cutadapt, Xenome, Bowtie, TopHat, SAMtools, Cufflinks
Application RNA-seq analysis
Organisms Heterocephalus glaber, Rattus norvegicus, Mus musculus
Diseases Neoplasms, Teratoma