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| Status |
Public on Jun 30, 2022 |
| Title |
ACS3 |
| Sample type |
SRA |
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| Source name |
Cortical neuron, alternative current stimulation,3μA 20hz 20min, 7days
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| Organism |
Rattus norvegicus |
| Characteristics |
tissue: embryo DIV 7d
cell line: primary cell
cell type: cortical neuron
genotype: WT
treatment: alternative current stimulation,3μA 20hz 20min，7days
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| Growth protocol |
Primary cortical neuron cultures were generated from embryonic day 16-18 Wistar rat embryos, neurons were plated at a density of 105 neurons/cm² on a 9.6 cm² six-well plates precoated with Poly-L-Lysine (Sigma–Aldrich, USA). Cultures were maintained in 2% B27-A, and 1% GlutaMAX (Gibco) supplemented with Neurobasal media (Invitrogen) until 14 d in vitro (DIV).
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using Trizol reagent following the manufacturer's procedure. The total RNA quantity and purity were analysis of Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit, high-quality RNA samples with RIN number > 7.0 were used to construct sequencing library.
After total RNA was extracted, mRNA was purified from total RNA (5ug) using Dynabeads Oligo (dT) (Thermo Fisher, CA, USA) with two rounds of purification. Following purification, the mRNA was fragmented into short fragments using divalent cations under elevated temperature (Magnesium RNA Fragmentation Module (NEB, cat.e6150, USA) under 94℃ 5-7min). Then the cleaved RNA fragments were reverse-transcribed to create the cDNA by SuperScript™ II Reverse Transcriptase (Invitrogen, cat. 1896649, USA), which were next used to synthesise U-labeled second-stranded DNAs with E. coli DNA polymerase I (NEB, cat.m0209, USA), RNase H (NEB, cat.m0297, USA) and dUTP Solution (Thermo Fisher, cat.R0133, USA). An A-base was then added to the blunt ends of each strand, preparing them for ligation to the indexed adapters, which including custom Unique Molecular Identifiers for minimizing sequence-dependent bias and amplification noise according to (Shiroguchi et al. 2012). Each adapter contained a T-base overhang for ligating the adapter to the A-tailed fragmented DNA. Dual-index adapters were ligated to the fragments, and size selection was performed with AMPureXP beads. After the heat-labile UDG enzyme (NEB, cat.m0280, USA) treatment of the U-labeled second-stranded DNAs, the ligated products were amplified with PCR by the following conditions: initial denaturation at 95℃ for 3 min; 8 cycles of denaturation at 98℃ for 15 sec, annealing at 60℃ for 15 sec, and extension at 72℃ for 30 sec; and then final extension at 72℃ for 5 min. The average insert size for the final cDNA librarys were 300±50 bp. At last, we performed the 2×150bp paired-end sequencing (PE150) on an Illumina Novaseq™ 6000 following the vendor's recommended protocol.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Reads obtained from the sequencing machines includes raw reads containing adapters or low quality bases which will affect the following assembly and analysis. Thus, to get high quality clean reads, reads were further filtered by Cutadapt (https://cutadapt.readthedocs.io/en/stable/,version:cutadapt-1.9)
We aligned reads of all samples to the research species reference genome using HISAT2 (https://daehwankimlab.github.io/hisat2/,version:hisat2-2.0.4) package, which initially remove a portion of the reads based on quality information accompanying each read and then maps the reads to the reference genome. And then by removing sequence-dependent bias and amplification noise using UMI-tools.
The mapped reads of each sample were assembled using StringTie (http://ccb.jhu.edu/software/stringtie/,version:stringtie-1.3.4d) with default parameters. Then, all transcriptomes from all samples were merged to reconstruct a comprehensive transcriptome using gffcompare software(http://ccb.jhu.edu/software/stringtie/gffcompare.shtml,version:gffcompare-0.9.8). After the final transcriptome was generated, StringTie and ballgown (http://www.bioconductor.org/packages/release/bioc/html/ballgown.html) were used to estimate the expression levels of all transcripts and perform expression abundance for mRNAs by calculating FPKM (fragment per kilobase of transcript per million mapped reads) value.
Genes differential expression analysis was performed by DESeq2 software between two different groups (and by edgeR between two samples). The genes with the parameter of false discovery rate (FDR) below 0.05 and absolute fold change ≥ 2 were considered differentially expressed genes. Differentially expressed genes were then subjected to enrichment analysis of GO functions and KEGG pathways.
Supplementary files format and content: fpkm and count
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| Submission date |
Jun 28, 2022 |
| Last update date |
Jun 30, 2022 |
| Contact name |
Mi Zhou |
| E-mail(s) |
mizhou0524@gmail.com
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| Phone |
+86 15823585001
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| Organization name |
Tianjin Medical University
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| Street address |
Anshan street 168th
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| City |
Tianjin |
| ZIP/Postal code |
31000 |
| Country |
China |
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| Platform ID |
GPL25947 |
| Series (1) |
| GSE207117 |
Electrical stimulation with a conductive ITO glass promotes neurite outgrowth and its putative mechanisms of action for direct and alternating current in primary cortical neurons |
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| Relations |
| BioSample |
SAMN29402639 |
| SRA |
SRX15930055 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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