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Sample GSM8192363

Query DataSets for GSM8192363

Status

Public on Jun 30, 2024

Title

astrocyte-24 h after injury-2

Sample type

SRA

Source name

brain

Organism

Drosophila melanogaster

Characteristics

tissue: brain
cell line: primary cell
cell type: astrocyte
genotype: WT
treatment: 24 h after injury

Treatment protocol

Construction and use of the HIT device were described previously (Katzenberger et al., 2013). Deflection of the spring to 90° was the standard angle for strike. Flies were collected at 3 days after eclosion and treated with TBI.

Growth protocol

Flies were maintained on standard cornmeal medium at 25°C otherwise indicated.

Extracted molecule

total RNA

Extraction protocol

Brains from adult flies (R86E01-Gal4/UAS-Redstinger) with no injury, 4 h after injury and 24 h after injury were dissected out in freshly prepared adult hemolymph saline (AHS) buffer, as previously described (Yang et al., 2016). Briefly, a total of ∼50 brains for each group was immediately transferred to an Eppendorf tube (1.5 mL) containing 200 µL AHS with Pronase (1 mg/mL; P5147, SigmaAldrich) and Dispase (1 mg/mL; LS02104, Worthington Biochemical Corporation). After tissue digestion for 30 min at 25°C, the enzyme medium was removed, followed by two brief, gentle washes with fresh AHS containing 2% PBS. Brain samples were then triturated by gently pipetting in 1 mL AHS through four fire-polished Pasteur pipettes with descending pore sizes (400 μm, 300 μm, 200 μm, and 100 μm) for a total of ∼20 min. Next, the dissociated cells were centrifuged for 10 min at 100 × g and the AHS was replaced with the same buffer to minimize any RNA content released from damaged cells during the trituration process. Finally, dissociated tissues were filtered through 45 μm filter and then put on ice. FACS sorting was performed according to the following gating procedure with 70 μm nozzle by BD FACS Aria: (1) an initial SSC-A/FSC-A gate to minimize debris; (2) a SSC-A/pe-Texa Ted-A gate to choose only red fluorescence positive cells. At this step, 1600 cells were sorted directly into ice-cold cell lysis buffer (oligo-dT30VN primer, ribonuclease inhibitor, dNTP mix) and stored at -80°C until all samples were ready for RNA extraction and cDNA amplification.
Cell samples were sent to the Anoroad Company for RNA extraction. An Oligo-dT primer was introduced to the reverse transcription reaction for first-strand cDNA synthesis, followed by PCR amplification to enrich the cDNA and magbeads purification step was used to purify the products. Briefly, Smart-Seq2 method was used to amplify cDNA. Concentration and integrity of cDNA was assessed using Qubit 3.0 Flurometer (Life Technologies) and Agilent 2100 Bioanalyzer (Agilent Technologies) to ensure the cDNA length was around 1-2 kbp. After quality control, 40 ng cDNA was used to be fragmented at 350 bp by Bioruptor® Sonication System (Diagenode Inc.). To construct Illumina library, end repair, 3’ ends A-tailing, adaptor ligation, PCR amplification and library validation were performed. PerkinElmer LabChip® GX Touch and Step OnePlus™ Real-Time PCR System were used for library quality inspection. Qualified libraries were loaded on Illumina Hiseq platform for PE150 sequencing.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

I24-8

Data processing

The remaining data was aligned against the UCSC D. melanogaster genome (dm6) using Hisat2 with the annotation file of the same version. On average, 92% of the initial reads were mapped to dm6. HTSeq was used to calculate the raw read counts of genes for each sample. DESeq2 was used to normalize the data. Transcripts were considered as significantly differentially expressed if their adjusted p values were less than 0.01 (p < 0.01) by the Benjamini-Hochberg method. Following normalization of the data, DESeq2 was used to perform pairwise comparisons between the control and experimental groups using parametric tests. Genes with absolute value of logarithmic fold change (log2FC) > 1.0 were qualified for the final set of DEGs. Volcano plots and heatmap were constructed for visualization of the resulting expression intensity data following the identification of DEGs and RNA-Seq data normalization by the adjusted p value calculations.
AnnotationHub 2.20.2 biomaRt 0.7 clusterProfiler 3.13 curl 4.3.2 DESeq2 1.30.1 DIOPT 8.0 dplyr 1.0.4 FastQC 0.11.7 ggplot2 3.3.5 ggrepel 0.9.1 org.Dm.eg.db 3.11.4 pheatmap 1.0.12 R 4.0.0 RSEM 1.3.1 RSeQC 2.5 STAR 2.6.0a Trimmomatic 0.39 VennDiagram 1.6.20
Assembly: dm6
Supplementary files format and content: tab-delimited text files include TPM values for each sample

Submission date

Apr 08, 2024

Last update date

Jun 30, 2024

Contact name

Tingting Li

E-mail(s)

li_tingting_29@sina.com

Organization name

Chinese Academy of Sciences

Street address

Beichen road