DOI: 10.57760/sciencedb.26421
title: U87 cell which overexpresdsed control vector or tau were used for RNA-Seq analysis
title: 采用RNA-Seq对过表达Tau或对照载体的U87细胞进行分析

description: This dataset originates from RNA sequencing analysis of human glioblastoma U87 cell lines, aiming to investigate the impact of Tau protein overexpression on the transcriptome. In this experiment, U87 cells were infected with either Tau-overexpressing plasmids or control plasmids (3 × 10⁶ cells per sample), yielding three independent samples for each group (Tau and control). Cells were lysed using TRIzol reagent, and total RNA was extracted. The concentration and purity of RNA were measured using the NanoDrop ND-1000 (NanoDrop, USA), and RNA integrity was assessed using the Agilent Bioanalyzer 2100, with all RIN values above 7.0, further confirmed by denaturing agarose gel electrophoresis.From each sample, 1 μg of total RNA was used for poly(A) RNA purification using Dynabeads Oligo (dT)25 (Thermo Fisher, USA) in two rounds. Fragmentation of the poly(A) RNA was then performed at 94°C for 5–7 minutes using the Magnesium RNA Fragmentation Module (NEB, USA). The fragmented RNA was reverse-transcribed into cDNA using SuperScript II reverse transcriptase (Invitrogen, USA), and the second-strand DNA was synthesized using E. coli DNA polymerase I, RNase H, and dUTP to produce U-labeled double-stranded DNA. After adenylation of 3' ends, sequencing adapters with T-overhangs were ligated to the A-tailed DNA fragments. Size selection was performed using AMPure XP magnetic beads, with an average insert size of 300 ± 50 bp. The U-labeled second-strand DNA was treated with UDG enzyme, and the libraries were amplified via PCR under the following conditions: 95°C for 3 min; 8 cycles of 98°C for 15 s, 60°C for 15 s, and 72°C for 30 s; followed by a final extension at 72°C for 5 min, generating the final sequencing libraries.Paired-end 150 bp sequencing (PE150) was performed using the Illumina NovaSeq™ 6000 platform, following the manufacturer's protocol. Raw sequencing data were processed using fastp to remove adapter contamination, low-quality bases, and reads with undetermined bases. Clean reads were aligned to the human reference genome (GRCh38) using HISAT2, followed by transcript assembly using StringTie. All transcriptomes were merged using gffcompare to generate a comprehensive transcript annotation. Gene expression levels were calculated by StringTie and normalized as FPKM (Fragments Per Kilobase of exon model per Million mapped reads). Differential gene expression analysis between groups was conducted using DESeq2, with differentially expressed genes (DEGs) defined as those with FDR &lt; 0.05 and |fold change| ≥ 2. edgeR was also applied in specific pairwise comparisons. Enrichment analysis of DEGs was conducted using the KEGG database.
description: This dataset originates from RNA sequencing analysis of human glioblastoma U87 cell lines, aiming to investigate the impact of Tau protein overexpression on the transcriptome. In this experiment, U87 cells were infected with either Tau-overexpressing plasmids or control plasmids (3 × 10⁶ cells per sample), yielding three independent samples for each group (Tau and control). Cells were lysed using TRIzol reagent, and total RNA was extracted. The concentration and purity of RNA were measured using the NanoDrop ND-1000 (NanoDrop, USA), and RNA integrity was assessed using the Agilent Bioanalyzer 2100, with all RIN values above 7.0, further confirmed by denaturing agarose gel electrophoresis.From each sample, 1 μg of total RNA was used for poly(A) RNA purification using Dynabeads Oligo (dT)25 (Thermo Fisher, USA) in two rounds. Fragmentation of the poly(A) RNA was then performed at 94°C for 5–7 minutes using the Magnesium RNA Fragmentation Module (NEB, USA). The fragmented RNA was reverse-transcribed into cDNA using SuperScript II reverse transcriptase (Invitrogen, USA), and the second-strand DNA was synthesized using E. coli DNA polymerase I, RNase H, and dUTP to produce U-labeled double-stranded DNA. After adenylation of 3' ends, sequencing adapters with T-overhangs were ligated to the A-tailed DNA fragments. Size selection was performed using AMPure XP magnetic beads, with an average insert size of 300 ± 50 bp. The U-labeled second-strand DNA was treated with UDG enzyme, and the libraries were amplified via PCR under the following conditions: 95°C for 3 min; 8 cycles of 98°C for 15 s, 60°C for 15 s, and 72°C for 30 s; followed by a final extension at 72°C for 5 min, generating the final sequencing libraries.Paired-end 150 bp sequencing (PE150) was performed using the Illumina NovaSeq™ 6000 platform, following the manufacturer's protocol. Raw sequencing data were processed using fastp to remove adapter contamination, low-quality bases, and reads with undetermined bases. Clean reads were aligned to the human reference genome (GRCh38) using HISAT2, followed by transcript assembly using StringTie. All transcriptomes were merged using gffcompare to generate a comprehensive transcript annotation. Gene expression levels were calculated by StringTie and normalized as FPKM (Fragments Per Kilobase of exon model per Million mapped reads). Differential gene expression analysis between groups was conducted using DESeq2, with differentially expressed genes (DEGs) defined as those with FDR &lt; 0.05 and |fold change| ≥ 2. edgeR was also applied in specific pairwise comparisons. Enrichment analysis of DEGs was conducted using the KEGG database.