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Next Generation Sequencing Facilitates Quantitative Analysis of control and HPK1-M46 Transgenic Mice Transcriptomes

ABSTRACT: Purpose: The goals of this study are to compare cerulean treated acinar cells of HPK1-M46 transgenic mice transcriptome profiling (RNA-seq) to study HPK1 pathway Methods: Total RNA was prepared from mouse pancreata harvested 24 hours after Cerulean treatment using RNeasy Mini/Micro Kit according to the manufacturer’s instructions. RNA quality was measured by the Agilent TapeStation 2200 (Agilent, Santa Clara, CA). RNA samples with RNA integrity numbers of ≥7 were processed for RNA-seq transcriptome analysis. Results: The RNA-seq libraries were pooled and sequenced, 83 bp single read, on Illumina NextSeq 550 or Illumina NovaSeq 6000 (Illumina, San Diego, CA). All sequencing data were demultiplexed and converted into fastq files by Illumina bcl2fastq2 v2.17.14. Filtered sequence reads were mapped to reference genome mm10 using STAR aligner with default settings. Conclusions: Our study represents the first detailed analysis of HPK1 transcriptomes, with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles.

ORGANISM(S): Mus musculus

PROVIDER: GSE223215 | GEO | 2023/06/21

REPOSITORIES: GEO

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Project description:Purpose: Pulmonary tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), has long been associated with the development of lung adenocarcinoma (LUAD) and sarcoidosis. These chronic lung diseases share histopathological similarities that challenge differential diagnosis. Methods: Human lung tissues were used for total RNA extraction with RNeasy Plus Mini Kit (Qiagen) according to the manufacturer’s protocol. RNA purity was assessed using a Nanodrop ND-2000 (Thermo Scientific), and each RNA sample had an A260:A280 ratio above 1.8 and an A260:A230 ratio above 2.0. RNA integrity was evaluated using the Agilent 2200 TapeStation (Agilent Technologies), and each sample had the RNA Integrity Number (RIN) above 7.0. The ribosomal RNAs (rRNAs) were removed using Ribo-Zero rRNA Removal Kits (Illumina). RNA libraries were then constructed by NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs) and evaluated using the Agilent 2200 TapeStation and Qubit® 2.0 (Life Technologies). Sequencing was performed by RiboBio Co., Ltd. on an Illumina HiSeq 3000 machine with paired-end 150-bp reads. The adaptors and low quality bases assessed using FASTQC 0.11.3 were trimmed by Trimmomatic 0.39 using the following options: TRAILING:20, MINLEN:235 and CROP:235. Trimmed reads were then aligned to the ensembl 79 (GRCh38.p2) reference genome using STAR 2.4.2a. FeatureCounts 1.6.2 was subsequently employed to convert aligned short reads into read counts for each sample. Genes with less than ten counts in two or more samples were removed. The data were then analyzed using R 3.4.4 and DEseq2 1.18.1. Differentially expressed genes of each disease group were identified using Wald statistics test, with fold-change > 2 and Benjamini–Hochberg (BH)-adjusted P < 0.1 as compared to NC group. Results: These findings provide novel pathogenic links and molecular markers for better understanding and differential diagnosis of pulmonary TB, LUAD and sarcoidosis.

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Next Generation Sequencing Facilitates Quantitative Analysis of control and HPK1-M46 Transgenic Mice Transcriptomes

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