melanoma and melanocytes sequencing

Description

We performed RNA deep sequencing on poly-A RNAs from three normal melanocyte primary cultures and three melanoma cell lines. The three melanoma cell-lines were recently established from lymph node metastases of melanoma patients. In addition, in terms of mutations of the raf pathway, one cell-line was not mutated (M383), one had Braf V600E mutation (M305) and the last one harbored N-ras Q61R mutation (M315). Our primary objective was to identify cancer specific neoantigens whose expression is controlled by an IRES-dependent translation. Initial selection of transcripts of interest was performed based on RNA-seq results presented here.

Steps to reproduce

First we selected melanoma tumor cell lines and primary melanocytes on which we performed total RNA extraction, quantitative and qualitative analysis (Qubit, RIN). Library preparation was performed using reagents from NewEnglandBiolab. Briefly polyA RNA was isolated with NEBNext Poly(A) mRNA magnetic isolation module, cDNA synthesis, adapter ligation and amplification was done using NEB Next Ultra II Directional RNA Library Prep Kit along with NEBNext Multiplex Oligos for Illumina. Single Read Sequencing was done with NextSeq500, Illumina. Typical read length was 75bp and sequencing was done at a depth of 40 million. Demultiplexing was done with bcl2fastq2.0 (Illumina), Fastq files passed quality control checks (per base sequence quality, per sequence quality scores, per base sequence content, GC content, N content, sequence length distribution, sequence duplication levels, overrepresented sequences, Kmer content). RNAseq data analysis was performed first by alignment on reference genome (GRCh38, release 91) using BowTie and TopHat package softwares. Reads quantification was done with Cufflinks and global transcriptomic file was generated using Stringtie. Experiment was performed by Helixio (Saint Beauzire, France)

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