Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Selection of RNA-seq library preparation protocol for a comprehensive transcriptome analysis in T acute lymphoblastic leukemia (T-ALL)

ABSTRACT: The aim of this study was to compare two different library preparation protocols: Illumina TruSeq Stranded mRNA (referred to as PA) and TruSeq Stranded Total RNA with Ribo-Zero Human/Mouse/Rat (RD) each with two different variants of the fragmentation step: standard fragmentation conditions (94°C for 8 minutes; further described as P1) and modified conditions (90°C for 2 minutes; further described as P2). The modification was expected to produce longer fragments. Each of the 5 tested T-ALL samples was sequenced using a combination of 4 different protocols: PA_P1, RD_P1, PA_P2, RD_P2 (for PA_P2 we performed two technical replicates). All 25 libraries were sequenced on Illumina NovaSeq6000 platform, in 2x150 mode, with a coverage of 150M reads/sample. High number of reads obtained for each of the samples allowed us to carry out a comprehensive comparison using various analysis methods aimed at identifying differentially expressed genes, alternative splicing events, gene fusions, somatic mutations and indels.

INSTRUMENT(S): Illumina NovaSeq 6000

ORGANISM(S): Homo sapiens

SUBMITTER: Małgorzata Dawidowska

PROVIDER: E-MTAB-10253 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

RNA-seq library preparation for comprehensive transcriptome analysis in cancer cells: The impact of insert size.

Jaksik Roman R Drobna-Śledzińska Monika M Dawidowska Małgorzata M

Genomics 20211103 6

With long reads and high coverage, RNA-seq enables comprehensive transcriptome analysis of cancer cells, provided that optimal length of libraries (and their inserts) is assured, to avoid overlap of paired reads and consequent loss of sequencing data. We assessed TruSeq Stranded library preparation protocols (poly(A) enrichment-PA and rRNA depletion-RD) for the thoroughness of transcriptome analysis of a heterogeneous cancer, acute lymphoblastic leukemia. We applied 2x150PE sequencing, >150 M re ...[more]

PMID: 34740778

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Project description:Abstract/Project overview Tumour recurrence can be influenced by the mutational status of a tumour but also how the tumour responds to therapy. Whilst activation of Type 1 interferon signalling is a hallmark of how cells respond to viral infection, in cancer cells, multiple stresses are known to activate this same response. In this study we have evaluated for the first time the changes in the interferon response induced by culturing prostate cancer cells under sphere-forming conditions and following irradiation. We identify that both stresses induced a selection/reprogramming towards a tumour-initiating/stem-like cell population That are known to be highly adaptable and have been shown to be resistant to multiple therapies. We report a conserved upregulated transcript profile for both conditions that is tightly associated with therapeutic resistance and cell survival in vitro and in vivo. The profile includes and is regulated by the Type 1 interferon master regulator IRF7, which when targeted delays tumour re-growth following irradiation. Understanding the impact of radiotherapy on the evolution of treatment resistant prostate cancer is critical for selecting effective treatment combinations. Our first-in-field data define irradiation as a selection pressure for a multi-treatment resistant, interferon response-dependent biology and further show that these cells acquire enhanced sensitivity to a combination of IKKε/TBK1 and MEK inhibition. Design of the sequencing experiment/Methods The RNA-seq experiment within this study was designed to identify radiation-responsive and IRF7-responsive genes in a prostate cancer cell-line, PC3. To do so we generated stable IRF7 knockdown derivatives using shRNA (referred to as 'IRF7' in the files) and scrambled controls (referred to as 'scr'). We subjected 5 million cells per condition to 1,2 or 3 doses of irradiation or mock irradiation with 24 hour or 72 hour recovery periods interspersed (annotated for example as '24-1', one dose with a 24-hour recovery period). At 24 hours or 72 hours after the final treatment we harvested cells and extracted total RNA using Trizol. To assess the yield and integrity of mRNA, 2ul of each sample was taken for analysis on a Fragment Analyzer Automated CE System (Advanced Analytical) and smear analysis run using a DNF-473 Standard Sensitivity NGS Fragment Analysis Kit (Advanced Analytical). Total RNA (1ug) was processed to first-stranded cDNA using the TruSeq Stranded mRNA Library Prep Kit (Illumina), following the manufacturer’s instructions for the low-throughput protocol. First-stranded cDNA samples were processed to barcoded cDNA libraries using a Diagenode IP-Star, according to the manufacturer’s instructions for Illumina TruSeq library prep. Resulting libraries were PCR-amplified to incorporate unique indices and cleanup performed using AMPure XP Beads (Becman Coulter) according to Truseq LT protocol. Resulting libraries were quantified, pooled and sequenced on a Nextseq 500 paired end 2x75Bp run. FASTQ files generated were de-multiplexed and adapter trimmed in the Illumina basespace and aligned to hg19 using Bowtie2 with default settings in the Basespace RNA-seq Express app. Resulting bam alignments files were exported and aligned to Refseq transcriptome annotation in Partek Genomic Suite and annotated transcripts with mapped sequence reads were quantified for relative expression in each sample and gene level RPKM (Reads Per Kilobase of transcript per Million mapped reads) counts generated for downstream analysis.

Dataset Information

Selection of RNA-seq library preparation protocol for a comprehensive transcriptome analysis in T acute lymphoblastic leukemia (T-ALL)

Publications

RNA-seq library preparation for comprehensive transcriptome analysis in cancer cells: The impact of insert size.

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