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Sample GSM8157272

Query DataSets for GSM8157272

Status

Public on Oct 03, 2024

Title

Aged01

Sample type

RNA

Source name

Bilateral Striatal Slice

Organism

Mus musculus

Characteristics

age: 18mo.
group: Aged

Extracted molecule

total RNA

Extraction protocol

mice were euthanized and brains were immediately placed in cold OCT. Embedded brains were then frozen at −20°C and transferred to −80°C for storage. Ten micrometer coronal slices of the striatum were sectioned and placed upon a functionalized coverslip covered with fluorescent beads.

Label

n/a

Label protocol

not applicable

Hybridization protocol

Once adhered to the coverslip the tissue was fixed (4% PFA in 1x PBS, 15 min, room temperature) followed by three washes with 1x PBS. After aspiration, 70% ethanol was added to permeabilize the tissue for 24 h. After a wash with Formamide Wash Buffer (30% formamide in 2x saline sodium citrate (SSC)), the sample was incubated with a custom MERFISH probe library (Vizgen) and left to hybridize for 48 h. The sample was then washed and incubated at 47 °C with formamide wash buffer twice, and then the tissue was embedded in a polyacrylamide gel followed by incubation with tissue clearing solution (2x SSC, 2% SDS, 0.5% v/v Triton X-100, and 1:100 proteinase K) overnight at 37°C. Then, the tissue was washed and hybridized for 15 min with the first hybridization buffer, which contained the readout probes associated with the first round of imaging. After washing, the coverslip was assembled into the imaging chamber and placed into the microscope for imaging.

Scan protocol

MERFISH imaging was performed on an automated Vizgen Alpha Instrument using imaging buffers, hybridization buffers and parameter files provided by Vizgen. Briefly, the sample was loaded into a flow chamber connected to the Vizgen Alpha Instrument. First, a low-resolution mosaic image was acquired (DAPI channel) with a low magnification objective (10X). Then the microscope was switched to a high magnification objective (60X) and seven 1.5 μm z-stack images of each field of view position were generated in 750 nm, 650 nm, and 560 nm channels. A single image of the fiducial beads on the surface of the coverslip was acquired and used as a spatial reference. After each round of imaging, the readout probes were extinguished, and the sample was hybridized with another set of readout probes.

Description

A panel of 383 genes for massively multiplexed FISH, for subcellular resolution for each gene of interest. Each cell has counts for each gene of interest

Data processing

. Raw data were decoded using the MERLIN pipeline (v.0.1.6, provided by Vizgen) with the codebook for the library. Each Id_
Values in the matrix show the genes (ID_Ref) and mean RNA expression. Individual raw files have transcripts and count for each cell for each gene in ID_Ref.

Submission date

Mar 20, 2024

Last update date

Oct 03, 2024

Contact name

Kay Linker

E-mail(s)

kaylinker@gmail.com

Organization name

UCLA

Department

Physiology