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Sample GSM5251852

Query DataSets for GSM5251852

Status

Public on Sep 22, 2022

Title

12829_hipp_cell

Sample type

SRA

Source name

hippocampus microglia

Organism

Sus scrofa

Characteristics

cell type: microglia
origin: hippocampus

Treatment protocol

Microglia and macrophages were FACs sorted. Samples were identified as microglia if they were both CD11bhigh/CD45-ve/low. Samples from the alveolar lavage were identified as macrophage if they were F4/80+ve/CD163low/-ve.

Extracted molecule

total RNA

Extraction protocol

For microglia and macrophages, cell suspensions were homogenised in 2ml lysing matrix D tubes (MP Biomedicals) on a FastPrep 24 at 6.5m/s for 50 seconds. After 5 minutes incubation at room temperature, an equal volume of chloroform was added to the homogenate, samples placed on a shaker at room temperature for 5 minutes, and then centrifuged at 12000rmp, 4°C for 15minutes. The 100μl of the resulting aqueous phase was transferred to a 96 well plate and 60μl isopropanol added. The plate was placed on an orbital shaker at medium speed for 1 minute. 20μl MagMaxTM beads were added to each well and plate returned to orbital shaker for 3 minutes. The 96 well plate was placed onto a magnetic separation rack and supernatant removed without disturbing the magnetic beads. This was repeated until all the aqueous phase had been used. The remaining extraction was performed as per the MagMaxTM -96 total RNA isolation manufacturers protocol, including a TURBOTM DNase clean up step, with a final total RNA elution volume of 40μl. For tissues, RNAlater stabilised tissues were homogenised in 1ml QIAzol reagent using a Qiagen TissueRuptor II on a medium speed setting for 40seconds, or until the lysate was uniformly homogeneous. Total RNA extraction was performed as per the Qiagen RNeasy Lipid tissue mini kit product guidelines, to a final elution volume of 40μl.
Takara SMARTer stranded total RNA-Seq vs2 library prep protocol.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Data processing

Reads quality filtered and trimmed with Bbtools.
Adaptor removed with Bbtools.
Genome alignment with Hisat2.
Counts generated using StringTie.
Genome_build: Sscrofa11.1
Supplementary_files_format_and_content: gene_count_matrix.csv: Gene count matrix, comma-separated file.

Submission date

Apr 18, 2021

Last update date

Sep 22, 2022

Contact name

Barbara Bo-Ju Shih

E-mail(s)

b.shih@lancaster.ac.uk

Organization name

Lancaster University

Department

Biomedical and Life Sciences