Computational protocol: TGF-β2-induced EMT is dampened by inhibition of autophagy and TNF-α treatment

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Protocol publication

[…] Briefly, for transcriptomic sequencing total RNA was isolated and taken for fragmentation and priming. The fragmented and primed mRNA was further subjected to first strand synthesis followed by second strand synthesis. The double stranded cDNA was purified using HighPrep PCR magnetic beads (Magbio Genomics Inc, USA). The purified cDNA was end-repaired, adenylated and ligated to Illumina multiplex barcode adapters as per NEBNext® Ultra™ Directional RNA Library Prep Kit protocol. The adapters used in the study were Illumina Universal Adapter: 5′-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3′ and Index Adapter: 5′-GATCGGAAGAGCACACGTCTGAACTCCAGTCAC ATCTCGTATGCCGTCTTCTGCTTG-3′. The adapter-ligated cDNA was purified using HighPrep beads and was subjected to Indexing-PCR to enrich. The final PCR product was purified with HighPrep beads. The sequencing library was initially quantified by Qubit fluorimeter (Thermo Fisher Scientific, USA) and its fragment size distribution was analyzed on Agilent Bio-analyzer. Finally, the sequencing library was quantified by qPCR using Kapa Library Quantification Kit (Kapa Biosystems, USA). The quantified libraries were pooled in equimolar amounts to create a final multiplexed library pool for sequencing on Illumina sequencer for 150bp paired-end reads. The cleaned reads were aligned to the reference genome using Tophat2. Transcript quantification was done using Cufflinks. Differential expression analysis was performed using Cuffdiff. Transcripts with log2fold change of 1 and above were considered as up regulated and those below -1 as down regulated. The raw reads were submitted to NCBI as BioProject PRJNA395629. Expression of some of the key genes regulating EMT was validated by RT-PCR, the conditions and primer sequences of which are described before. […]

Pipeline specifications

Software tools TopHat, Cufflinks
Application RNA-seq analysis
Diseases Carcinoma, Hepatocellular, Neoplasms
Chemicals Oxygen