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Sample GSM4151319 Query DataSets for GSM4151319
Status Public on Oct 04, 2022
Title S1006
Sample type SRA
Source name Hippo Pyramidal neruon
Organism Mus musculus
Characteristics strain: C57BL/6
tissue: brain
cell type: Hippo Pyramidal neruon
Treatment protocol Three different doses (2.88×10^8, 2.88×10^9, 2.88×10^10 vector genomes) of rAAV5 vectors carrying cre-dependent mCherry mRNA (rAAV5-DIO-mCherry) were chosen in this study, and the lower rAAV5 was used as a default control. To isolate the fluorescent cells for transcriptional profiling, slices of the hippocampus were sectioned three weeks after rAAV5-DIO-mCherry injection, and a single-cell suspension was generated.
Extracted molecule total RNA
Extraction protocol Mice were perfused with artificial cerebrospinal fluid under anesthesia state which may cool the brain more rapidly(Zeisel et al., 2018). We slightly modified their protocol. ACSF consisting of 87 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 26 mM NaHCO3, 75 mM sucrose, 20 mM glucose, 1 mM CaCl2, 7 mM MgSO4 and 0.1 µM tetrodotoxin was equilibrated in 95% O2 /5% CO2 before use. Mice were anesthetized with 1% pentobarbital sodium, followed by transcardial perfusion through the left ventricle with ice-cold ACSF. The brain was removed, and 300-μm vibratome sections were collected using Vibroslice (VT 1000S; Leica) in ice-cold ACSF. The hippocampus was microdissected and transferred to 100-mm dishes containing 7 mL digestion solution (2 mg/mL papain, Worthington, LS003119; 0.5 mM EDTA-2Na; 2.5 mL/L Glutamax 100X, ThermoFisher; resolved in Hibernate A, ThermoFisher) with 30-40 min enzymatic digestion at 30 ℃. The digestion solution was weakly acidic after the addition of papain, and 300-μL neruobasal A (ThermoFisher, 10888022) was required to adjust the pH to 7.4. After incubation, the tissue was transferred to a 15-mL tube containing 5 mL ice-cold HABG (60 mL Hibernate A; 20 mL/L B27 50X, ThermoFisher; 2.5 mL/L Glutamax 100X). The tissue pieces were gently triturated through Pasteur pipettes with polished tip openings of 0.6 mm, 0.3 mm and 0.15 mm diameter. The supernatant was transferred to a 15-mL tube after settling for 1 min. The sediment was resuspended in 2 mL HABG, and the supernatant was transferred. The procedures were repeated until the sediment was almost dissociated. The cells were enriched by centrifugation (800 g, 5 min, 4 ℃), resuspended in 1 mL HABG containing 1 mg/mL DNase I (Roche, 11284932001), and filtered with a 40-μm cell strainer (Falcon). To exclude dead cells, 0.1 μg/mL DAPI (Invitrogen) was added to the single-cell suspension. Then, the cells were immediately loaded into the Beckman MoFlo XDP Cell Sorter system for fluorescence-activated cell sorting (FACS). The high mCherry and low DAPI fluorescent cells were isolated into 1.5-mL tubes containing 5.4 µL sample buffer, 0.6 µL RNase inhibitor and 4 µL RNase-free water (Discover-sc WTA Kit V2, N711, Vazyme). Oxygenation and a short time of dissection were crucial to keep a high rate of survival in the cell suspension. For cell sorting experiments, ∼500 - 1,000 cells were isolated from 2 - 3 mice, and this represented 1 biological sample in our analysis. For each group, 3-5 biological replicates were obtained.
RNA libraries were prepared for sequencing using standard Illumina protocols
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model HiSeq X Ten
Description CamkⅡα- cre(C57BL/6)
Data processing Base-calling performed using Bcl2fastq (v2.17.1.14)
To remove technical sequences from raw data, including adapters, polymerase chain reaction (PCR) primers, or fragments thereof and quality of bases lower than 20, raw data in the fastq format were processed by Trimmomatic (v 0.30) to obtain high quality reads(Bolger et al., 2014). All libraries were aligned to UCSC mm10 using Hisat2 (v 2.0.1)(Kim et al., 2015). The clean data were monitored by quality control (QC) criteria. QC-qualified samples were included for each group (Q30 > 85%, total mapped > 70%). Raw counts of the number of tags on each gene were generated by using HTSeq (v 0.6.1), and the Fragments Per Kilobase of exon model per Million reads mapped (FPKM) is a normalized estimation of gene expression based on read ccount.
Genome_build: UCSCmm10
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each Sample ...
Submission date Nov 06, 2019
Last update date Oct 04, 2022
Contact name YISI LIU
Street address SHATAI ROAD
ZIP/Postal code 510000
Country China
Platform ID GPL21273
Series (1)
GSE139997 Effects of Different Doses of rAAV 5-mCherry on Transcriptome of Hippocampal Pyramidal Neurons in Male Mouse
BioSample SAMN13221172
SRA SRX7106567

Supplementary file Size Download File type/resource
GSM4151319_M3.txt.gz 236.5 Kb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record
Processed data provided as supplementary file

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