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Sample GSM3457273 Query DataSets for GSM3457273
Status Public on Aug 26, 2019
Title P28 WT Astrocyte rep 5
Sample type SRA
Source name Acutely isolated cortical astrocytes
Organism Mus musculus
Characteristics strain: C57BL/6
cell type: Astrocyte
age: Postnatal days 25-31
genotype: WT
Sex: Male
Extracted molecule total RNA
Extraction protocol Astrocytes were acutely isolated as previously described (Holt and Olsen, 2016; Kahanovitch et al., 2018; Stoica et al., 2017). Following dissociation, microglia and myelin were first removed from the cell suspension. Cells were incubated for 15 minutes at 4°C with 15µL of Miltenyi Biotec’s Myelin Removal Kit and Cd11b+ MicroBeads. The suspension was then applied to a prepped LS column, washed three times, and the flow-through collected. This flow through was subsequently used to isolate astrocytes utilizing Miltenyi Biotec’s ACSA-2+ MicroBead kit. The cell suspension (in 150uL 0.5% fatty-acid free BSA in PBS) was incubated at 4°C for 15 minutes with 15-20µL FcR blocker, followed by a 15 minute incubation with 15-20µL ACSA-2 microbeads. Cells were applied to a prepped LS column. Astrocytes were eluted from the LS column after three washes, with 5mL buffer and the supplied plunger. Total RNA was isolated using Ambion’s PureLink RNA Mini Isolation kit according to the manufacturer’s instructions. RNA samples designated for RNA Sequencing were eluted in 30 µL filtered, autoclaved Mill-Q water. RNA samples were tested for quality on the Agilent Tapestation 2200 (Agilent Technologies, Santa Clara, CA).
The NEB Next rRNA Depletion Kit (NEB #E6310X) was used to process 250 ng of total RNA. RNA-Seq libraries (400 bp) were created using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB #E7760L). Samples were individually indexed using the NEBNext Multiplex Oligos for Illumina (NEB #E6609S). Adapter ligated DNA was amplified in 13 cycles of PCR enrichment. Libraries were quantified with the Quanti-iT dsDNA HS Kit (Invitrogen) and qPCR. Library validation was performed on the Agilent 2200 Tapestation. Independently indexed stranded cDNA libraries were pooled and sequenced for 150 cycles with the Illumina NovaSeq 6000 S2 Kit. All samples were sequenced at 85-90 million read depth, paired-end 2 x 150 bp, and in reverse-stranded orientation.
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NovaSeq 6000
Data processing Raw RNA-Seq reads were concatenated (per R1 and R2 fastq read, respectively) and quality trimmed using Trim Galore! Version 0.4.3 . Sequence quality of trimmed reads was inspected using FastQC (version 0.11.15) .
The STAR aligner (version 2.5.2) was used in the basic two-pass mode to align the trimmed reads to the iGenomes UCSC mm10 mouse genome . BAM files were sorted by coordinate, and indexed using SAMtools (version 1.3.1).
STAR-aligned BAM files were processed in the University of Alabama at Birmingham Galaxy platform using Stringtie (Galaxy tool version as described in the recommended workflow , with minor modifications: 1) the reverse strand option was selected and 2) the iGenomes UCSC mm10 genome was used as the reference guide assembly data set for the first Stringtie run.
Genome_build: mm10
Supplementary_files_format_and_content: Tabular file containing StringTie output assembled transcript expression
Submission date Nov 05, 2018
Last update date Aug 28, 2019
Contact name Michelle L. Olsen
Phone (540) 231-7394
Organization name Virginia Polytechnic and State University
Department School of Neuroscience
Lab LS1 RM 213(540)
Street address 970 Washington Street SW
City Blacksburg
State/province VA
ZIP/Postal code 24060
Country USA
Platform ID GPL24247
Series (1)
GSE122176 Astrocyte morphogenesis is dependent on BDNF signaling via astrocytic TrkB.T1
BioSample SAMN10380322
SRA SRX4979937

Supplementary file Size Download File type/resource
GSM3457273_StringTie_P28_WT_A5_transcript-level_expression.tabular.txt.gz 1.3 Mb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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