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Transcriptome profiling of human glioma and normal brain tissues by rRNA-deleted total RNAseq

ABSTRACT: Purpose: To compare the coding and non-coding transcriptomic changes in human gliomas compared with normal brain tissues. Methods: Biopsies were collected from 85 adult glioblastomas, 18 lower grade gliomas, and 15 normal brain tissues. Total RNA was isolated using the Qiagen RNeasy Mini kit or Trizol reagent according to the manufacturer's instruction. Stranded, rRNA-deleted libraries were prepared using the Illumina stranded Total RNA TruSEQ kit according to the manufacturer's instruction. Sequencing was performed on Illumina HiSeq 4000 instrument according to the manufacturer instructions. Reads were aligned to the human genome GRCh38 using HISAT2 (Version 2.1.0). Read counts for each gene were quantified using HTSeq-count (Version 0.10.0) and then normalized by DESeq2 (r package, version 1.20.0). Results: Approximately 20 million paired-end reads at the length of 50 bp were obtained from each sample. The genome mapping efficiency was 85% on average for all samples. Altered expression of 6,132 protein-coding genes and 1,270 lncRNAs were identified. Conclusions:We report the coding and non-coding transcriptomic changes in human gliomas compared with normal brain tissues.

ORGANISM(S): Homo sapiens

PROVIDER: GSE147352 | GEO | 2021/01/26

REPOSITORIES: GEO

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Project description:Purpose: The aim of this study is (1) to identify the chromatin occupancy of the epigenetic regulator Smchd1 in neural stem cells (NSCs) derived from E14.5 mouse brain; (2) to profile key epigenetic marks H3K4me3, H3K27me3 and DNA methylation in wild type and Smchd1 null NSCs; (3) to identify the chromatin occupancy of Ctcf in wild type and Smchd1 null NSCs. Methods: Chromatin immunoprecipitation for Smchd1, H3K4me3, H3K27me3 and Ctcf was performed essentially as in (Nelson et al. 2006). Briefly, nuclei were isolated from formaldehyde crosslinked NSCs and chromatin was fragmented by sonication. Chromatin immunoprecipitation was performed with corresponding antibodies for Smchd1, H3K4me3 and H3K27me3. DNA was extracted from the immunoprecipitated fraction following reverse-crosslinking. Isolated DNA was used to generate sequencing libraries with Illumina's TruSeq DNA Sample Preparation Kit or Ovation Ultralow system (NuGen) according to manufacturer's instruction. Libraries were pooled and sequenced on the Illumina HiSeq 2000 platform for 100 bp single-end reads. Image analysis was performed in real time by the HiSeq Control Software (HCS) v1.4.8 and Real Time Analysis (RTA) v1.12.4.2, running on the instrument computer. Real-time base calling on the HiSeq instrument computer was performed with the RTA software. Illumina CASAVA1.8 pipeline was used to generate the sequence data. To examine the level of DNA methylation, genomic DNA was extracted using an AllPrep DNA/RNA Mini Kit (Qiagen) and methylated DNA was isolated via binding to the methyl-CpG binding domain of human MBD2 protein coupled beads using the MethylMiner methylated DNA enrichment kit (Life Technologies) according to the manufacturer’s instructions. Isolated DNA was used to generate sequencing libraries as for the ChIP-seq experiment with Illumina’s TruSeq DNA Sample Preparation Kit according to manufacturer's instruction and sequenced on the Illumina HiSeq 2000 platform for 49 bp single-end reads. Sequencing analysis was performed as described for the ChIP-seq experiments.

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Transcriptome profiling of human glioma and normal brain tissues by rRNA-deleted total RNAseq

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